CN104736721B - The molecular labeling and its application method of the low palmitic acid content of sunflower (Helianthus annuus) - Google Patents

Abstract

This disclosure relates to for identifying the method and composition of the sunflower plants with low palmitic acid content phenotype.Some embodiments are related to the molecular labeling of plant and germplasm (germplasm) for identifying, selecting, and/or constructing low palmitic acid content, or for identifying and the molecular labeling of the plant of counter-selection (counter-select) relatively high palmitic acid content.The sunflower plants with low palmitic acid content phenotype that present disclosure also relates to be generated by the method using at least one label described herein.

Description

The molecular labeling of the low palmitic acid content of sunflower (Helianthus annuus) and its Application method

Prioity claim

Patent application claims obtained the interests of U.S. Provisional Patent Application Serial number 61/613,383, in 2012 3 The moon is filed an application on the 20th.

Technical field

This disclosure relates to for identify the composition and method with the sunflower plants of low palmitic acid content, the wherein party Method is with molecular genetic marker identification, selects and/or construct low palmitic acid content plant.Present disclosure also relates to sides through the invention The sunflower plants for the low palmitic acid content of display that method generates.

Background

Cultivating sunflower (Helianthus annuus L.) is a main source of whole world vegetable oil.In the U.S., About 4,000,000 acres of sunflowers of plantation every year, are predominantly located at Dakota (Dakotas) and the Minnesota State (Minnesota).

In past 10 years, the sunflower acreage of U.S.'s plantation quickly expands, this was partly due to day Several impressive progresses in certain herbaceous plants with big flowers breeding and breed improvement field, the discovery including cytoplasmic male sterility and restoring gene. This discovery allows to generate hybrid sunflower.The hybrid so generated is introduced into early stage the 1970s.About to day Fick is shown in the description of certain herbaceous plants with big flowers cytoplasmic male sterility (CMS) and hereditary fertility restorer, " Breeding and Genetics ", " to day Certain herbaceous plants with big flowers science and technology " (Sunflower Science and Technology) the 279-338 pages (J.F.Carter is edited .1978)。

Sunflower oil mainly includes palmitinic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and Asia The fatty acid such as numb acid (18:3).Although there is also other not common fatty acids in plant, palmitinic acid, stearic acid, oleic acid, Linoleic acid plus linolenic acid accounts for about 88% of fatty acid present in cosmopolitian plant oil yield.J.L.Harwood,"Plant Acyl Lipids:Structure,Distribution and Analysis,”4Lipids:Structure and Function, P.K.Stumpf and E.E.Conn edit (1988).Palmitinic acid and stearic acid are saturated fatty acids, in certain researchs In have been proven that they facilitate the increase of blood plasma cholesterol level, this increase leads to one that coronary heart disease develops Factor.In the light of recent researches, the high vegetable oil of unsaturated fatty acid (such as oleic acid and linoleic acid) content may have reduction blood Starch the ability of cholesterol.

In general, saturated fatty acid also has fusing point more higher than same carbon number purpose unsaturated fatty acid, this facilitates Cold resistance (cold tolerance) problem of food, and wax sense of the food in mouth during intake can be further contributed to (waxy) or greasy feeling.Also it is known that with saturated fatty acid lower than about 3% fat and oil made of food usually everyone Part (serving) contains the saturated fat less than 0.5 gram, therefore in current label supervisory system (labeling Regulation it can be marked as under) containing " zero saturated fat ".

The exploitation of any new, ideal plant germplasm all includes many steps.Plant breeding procedures will from two or Multiple cultivars or anticipant character from extensive various source are combined into breeding pond (breeding pool), by selfing and Desired phenotype is selected to develop cultivar from breeding pond.New cultivar is assessed, to determine which has business potential. The problem of plant breeding is analysis first and determines existing germplasm and disadvantage establish project objective, and define specifically breeding mesh 's.It is the germplasm that selection has the character for meeting project objective in next step.Target is that composition comes from parent's kind in single variety The improved combination of the anticipant character of matter.These important characters may include higher seed production, resist to disease and insect Property, better stem and root, to the tolerance and better agronomy quality of arid and heat.

The selection of breeding or selection method depends on the mode of plant propagation, the heredity of modified character and commercially Type (such as the F of the cultivar used₁Hybrid cultivar, pure lines cultivar etc.).Character heritable for height, selection exist The superior plant individual for solely putting assessment may be effective, and the character low for heredity, should be based on to the plant of nearly edge The resulting average value of the repeat assessment of the family of object is selected.Popular selection method generally includes kind of a pedigree selection, improvement Pedigree selection, mixing selection (mass selection) and recurrent selection (recurrent selection).

The complexity of heredity influences the selection of breeding method.Back cross breeding is used for one or a few can lose height The advantageous gene transfer of the character of biography is into ideal cultivar.This method is widely used for the cultivation of breeding disease resistance Kind.A variety of recurrent selection techniques have been used for improveing the quantitative inheritance character controlled by multiple genes.Recurrent selection is in self-pollination Use in crop depends on the easness of pollination, the frequency for successful cross of pollinating every time and from the miscellaneous of each successful cross Hand over the number of offspring.

Each breeding project should include the periodical objective evaluation to selection efficiency.Evaluation criteria with target and Purpose and change, but should include that the annual gain (gain from selection) from selection (is based on and suitable mark Quasi- comparison), total value and the per unit investment of advanced breeding system (advanced breeding line) (such as annual, flower Each dollar (each pound) taken etc.) generate successful cultivar number.Then, promising advanced breeding system is carried out thorough The test at bottom, and be compared 3 years or more with the appropriate criteria product in the environment for representing business goal area.From optimal system Select the candidate of new commercial cultivar；Those still have insufficient system to can be used as parent to produce in a few character The raw new group for further selection.These processes are eventually leading to listing (marketing) and sell (distribution) The step of, it is counted from the time for carrying out first time hybridization and generally takes 8-12.Therefore, the exploitation of new cultivar is time-consuming mistake Journey needs accurately to preplan, the direction change of the effective use of resource and bottom line.

Most difficult task is to identify genetically superior individual in plant breeding.Identifying a kind of method of superior plant is Observe the performance (performance) for the standard cultivar that it is planted relative to other experimental plants and extensively.If single is seen No conclusion is examined, then repeated observation can provide the more preferable assessment to its genetic worth.This task is extremely difficult, because (for For most of characters) true genotypic value covered by other plant traits mixed or environmental factor.

The purpose of sunflower plants breeding is to develop new, unique and superior sunflower cultivar and hybrid. Breeder initially selects and hybridizes two or more parental departments, then carries out duplicate selfing and selection, generates many new Genetic make up.Theoretically, breeder can generate billions of kinds of different genetic make ups by hybridization, selfing and mutagenesis.In this way Breeder there is no directly controlling on cellular level to the process.Therefore, two breeders will not develop with identical The same tie of sunflower character, or even very similar be.

Every year, plant breeder selects germplasm to be advanced to next generation.In unique and different geography, weather and soil Under the conditions of plant the germplasm.Then, during growth period and at the end of further selected.The cultivar developed is not It is predictable.This unpredictability is due to caused by the selection of breeder, which occurs in unique environment, and can not It is controlled on DNA level (using conventional breeding method), and millions of kinds of different possible hereditary groups can be generated It closes.The unpredictable final gained strain that he is developed of breeder with ordinary skill, at most may be with very thick Slightly and the mode of summary is predicted.Similarly, same breeder is not available identical initial parents and identical choosing It selects technology and generates identical cultivar twice.This unpredictability causes the exploitation of superior novel facing sun certain herbaceous plants with big flowers cultivar to spend Take a large amount of resource, money etc..

The exploitation of new sunflower cultivar requires exploitation and selection Sunflower Varieties, hybridizes these kinds and select excellent Hybrids more.By in the selected male artificial hybridization that can be educated between parent or by using male sterility system To generate hybrid seed.Selecting to can be shown that seed really to these hybrids is certain monogenic characters of hybrid (such as pod Color, the color of flower, the color and Herbicid resistant of pubescence (pubescence)).About parental line and hybrid The data influence breeder of phenotype decides whether to continue specific hybrids.

Pedigree breeding is commonly used in the crop of improvement self-pollination.In pedigree breeding, two kinds are had advantageous, complementary The parents of character are to generate F₁Offspring.It is one or several from F by making₁The plant selfing of offspring generates F₂Group.To most The selection of good individual can be from F₂Start in group；Then, from F₃Middle beginning selects the optimized individual in best family.In order to mention Height, can be in F to the effectiveness of selection with low genetic character₄Start to carry out retest to family in generation.In breeding Later period (such as F₆And F₇), to optimal system or the mixture for being with similar phenotype is tested, and is investigated as new cultivation The release potentiality of kind.Mixing and recurrent selection can be used to improve the group of self-pollination or cross pollinated plant.By making Several difference parents mutually hand over to identify or create the variable heterozygous individual group of heredity.Based on individual superiority, offspring outstanding, Or superior combination ability selects optimal plant.Hand over the plant selected mutually to generate new group, it wherein can be after Continuous further selection circulation.

Back cross breeding has been used for simple and the heritable character of height gene transfer to desired homozygous cultivation Cultivate or inbred strais in, such homozygous cultivar or inbred strais are recurrent parents.The character source to be shifted is referred to as " donor parent This ".The expected attribute with recurrent parent (such as cultivar) of resulting plant and the anticipant character shifted from donor parents.Just Begin to select the individual with donor parents phenotype, and repeat to hybridize (backcrossing) with recurrent parent after hybridization.Resulting plant is expected Attribute with recurrent parent (such as cultivar) and the anticipant character from donor parents transfer.

In sunflower breeding, " simple grain transmission method " (single-seed descent procedure) refers to plantation point It peels off, then from every plant of gained one seed sample of plant harvest, and uses and harvest under the plantation of this seed sample all one's life Generation.When group is from F₂When generation is advanced into desired inbreeding level, the plant in strain institute source can respectively date back different F₂ Individual.In the number of plant per generation, all declines in group, this is because some seeds cannot sprout or some plants cannot generate Even a seed.As a result, when completing generation advance, not all F initially sampled in group₂Plant all can be by rear Table from generation to generation.

In a variety of submethods (multiple-seed procedure), sunflower breeder is usually from every in group Strain plant harvest seed, and make their threshings together to form large quantities of (bulk).It is planted using large quantities of a part Next generation, and a part is saved.This method is referred to as the simple grain transmission method improved.A variety of submethods are used to save and receive Labour when obtaining.It is more faster than removing from every plant a seed in simple grain transmission method with hand that seed is removed with machine.More seeds Method also makes it is likely that planting equal number of group's seed from generation to generation for each inbreeding.Sufficient seed is harvested to compensate Those do not sprout or do not produce seed bearing plant.

Suitable test should detect any grave error, and it is superior compared to existing cultivar to establish new cultivar Or improvement is horizontal.In addition to showing superior performance, it should need to meet the new cultivation that industry standard is compatible or taps new markets It cultivates.Test before releasing new cultivar is contemplated that the technological merit of research and development cost and final cultivar.By In the change of advertisement and marketing, seed and the commercial production practices specially needed and the utilization of new product, the introducing of new cultivar May and so that seed producers, grower, processor and consumer is born additional cost.For the cultivar of seminal propagation, It must be feasible for easily and economically generating seed.

The target of plant breeder is that selection plant and enrichment have anticipant character, such as reduced palmitic acid content, The plant population of individual, eventually leads to the raising of agricultural productivity.Consistent with the above, the persistent goal of sunflower breeder is out Send out stabilizations, high yield, cultivar firm on agronomy.Current target include the grain amount for making to produce on soil used, with And the food supply of animals and humans is maximized.In order to realize these purposes, sunflower breeder must select and develop tool There are the sunflower plants for the character that can produce superior cultivar, and realizes it in such a way that cost efficiency is highest.Molecular labeling can To use during marker assisted selection (MAS), help to identify and select individual with the genetic property chain with label or The family of individual.

It is open

Chain molecular labeling can be used to the label of low palmitic acid content character in easyization sunflower with low palmitic acid content Assisted Selection.Compared with palmitic acid content parting (phenotyping), marker assisted selection is in time, cost and labour side Face can provide significant advantage.Disclosed herein is in the identified region low palmitic acid content QTL positioned at sunflower genome or Specific markers near it, these labels are polymorphism in parent genotype, and with the low chain (example of palmitic acid content phenotype Such as close linkage).These labels mention in the marker assisted selection of sunflower plants and cultivar with low palmitic acid content Superior effectiveness is supplied.

The first sunflower plants of low palmitic acid content are shown this document describes identification or are included in this sunflower plants The method of interior germplasm.In some instances, it is contained to show that the first sunflower plants of low palmitic acid content or germplasm can be The plant of palmitic acid content low (reducing) more observed than in the mother plant or germplasm of first plant or germplasm or kind Matter.In some instances, show that the first sunflower plants of low palmitic acid content or germplasm can be contained palmitic acid content ratio Observed by being belonged in the specific conventional plant or germplasm of same species (such as sunflower) with first plant or germplasm The plant of low (reducing) or germplasm.Some embodiments of these methods may include detecting first sunflower plants or kind At least one label chain with low palmitic acid content in matter, wherein at least one label is selected from the group: HA0031B； HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870；HA0907；HA0612A；And and HA0031B, At least one of HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, and HA0612A are chain The label of (such as close linkage).

Also describe the method for generating the sunflower plants or germplasm with low palmitic acid content.Some realities of these methods The scheme of applying may include seeping at least one label chain with low palmitic acid content from the first sunflower plants or germplasm gene Enter into the second sunflower plants or germplasm, to generate the sunflower plants or germplasm that are likely to that there is low palmitic acid content.? In these examples, which is selected from the group: HA0031B；HA0908；HA1665；HA0304A；HA0850； HA0743；HA0870；HA0907；HA0612A；And and HA0031B, HA0908, HA1665, HA0304A, HA0850, Any of HA0743, HA0870, HA0907, and HA0612A chain label.In some specific embodiments, also Include the sunflower plants or germplasm generated by preceding method.

Some embodiments include the method for generating transgenosis sunflower plants.The example of these methods may include One or more exogenous nucleic acid molecules are introduced into target sunflower plants or its offspring, wherein the one or more external source core At least one of acid molecule includes to mark chain sunflower genome nucleotide sequence at least one, it is described at least one It marks chain with low palmitic acid content；Or wherein at least one of the one or more exogenous nucleic acid molecule include can be with The nucleotide sequence of nucleotide sequence specific hybrid as described below: the nucleotide sequence and at least one label are chain, should At least one label is chain with low palmitic acid content.Chain label can be selected from the group with low palmitic acid content: HA0031B； HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870；HA0907；HA0612A, and and HA0031B, Any of HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, and HA0612A chain mark Note.In some instances, for generating transgenosis sunflower plants, gained transgenosis sunflower plants may include preceding method Low palmitic acid content.

Some embodiments include for identify may comprising low palmitic acid content sunflower plants system and reagent Box.The specific example of these systems and kit may include a series of nucleic acid probes, and each includes can be with following institute The nucleotide sequence for the nucleotide sequence specific hybrid stated: the nucleotides sequence is listed in sunflower to be connected at least one label Lock, the label are chain with low palmitic acid content.The label chain with low palmitic acid content can be selected from the group in sunflower: HA0031B；HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870；HA0907；HA0612A, Yi Jiyu Any of HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, and HA0612A Chain label.For identify may comprising low palmitic acid content sunflower plants system and kit specific example also It may include detector, be configured to detection one or more and exported from the signal of the series nucleic acid probe or its amplicon, The existence or non-existence of at least one label chain with low palmitic acid content is identified whereby.Specific example include by this extremely The existence or non-existence of a few label reduces associated instruction with palmitic acid content.

Brief description

Fig. 1 includes a width GC-FID FAME chromatogram, is shown by being compared with the retention time of methyl ester reference standard Compared with and identify palmitinic acid methyl ester peak.Based on total mark peak area, calculated individually for all analytes in reference standard Percentage area.A heptane blank sample is also injected, for identifying any pollution on GC.

Fig. 2 includes a width graphical display, shows the distribution of the palmitic acid content of 23,040 sample.The value of the distribution is described It is provided in following table 1-2.

Fig. 3 includes 384 obtained by an excellent Sunflower Lines and an incross with low palmitic acid content The F of individual₂The histogram of the palmitic acid content of group.

Fig. 4 includes the schematic diagram of a low palmitic acid content oligogene seat in sunflower in linkage group 5 (LG5).? Identified several SSR markers and described locus close linkage or in its flank.

Fig. 5 includes schematically showing for the chain situation between low palmitic acid content locus and several SSR markers, display The position of main low palmitinic acid QTL on LG5.LDO score is shown on Y- axis, shows label and base on x- axis as unit of cM Because of the distance of seat.LOD score be by using Map QTL software program (J.W.Van Ooijen, M.P.Boer, R.C.Jansen,C.Maliepaard(2002)Map QTL 4.0:software for the calculation of QTL positions on genetic maps,Plant Research International,Wageningen,The Netherlands) agreement (multiple interval protocol) determination between the multi-region realized.

Implement mode of the invention

I. several embodiment general views

There are many reason expectations to generate the certain herbaceous plants with big flowers with low palmitinic acid and stearic acid level and high oleic acid and linoleic acid level Flower seed oil.Embodiment of the present invention includes, for example, for identifying the sunflower plants containing low palmitic acid content and/or taking Band is predictable and determines the composition and method of the germplasm of the genotype of low palmitinic acid phenotype.In some embodiments, include The method for making these sunflower plants and germplasm.These methods may include, such as but be not limited only to, desired low palmitinic acid The gene transgression and/or genetic transforming method of content marker allele.It include passing through these methods in specific embodiment The sunflower plants and/or germplasm of production, for example, it is previously described.For selecting the sunflower plants containing low palmitic acid content And/or the system and kit of the germplasm of the genotype of the predictable and determining low palmitinic acid phenotype of carrying are also certain embodiments Feature.

Identify and select the sunflower plants containing low palmitic acid content to be capable of providing using MAS efficient and environmental-friendly Generate the method with the plant of desired oil content.Embodiment of the present invention provide several sunflower marked locus and QTL chromosome interval (QTL chromosome interval), they show being total to statistically significantly with low palmitic acid content It separates (therefore can predict and determine low palmitic acid content).To these labels, or it is chain with these labels therefore therewith etc. The detection of the additional gene loci of valence can be used for marking the auxiliary sunflower procedure of breeding, to generate low palmitic acid content plant and kind Matter.

Some embodiments provide for identify show low palmitic acid content the first sunflower plants or germplasm (such as Strain or kind) method.In some embodiments, one or more and low palm fibre is detected in the first sunflower plants or germplasm At least one equipotential of the marked locus (such as multiple marked locus) of (such as the close linkage) of the palmitic acid acid linkage of characters Gene.Marked locus can be selected from the locus of Fig. 4, comprising: HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and chain other labels are marked at least one aforementioned QTL.

In some instances, multiple marked locus can be selected or identified in identical plant or germplasm.For example, wanting The multiple marked locus for selecting or identifying in plant or germplasm may include HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and mark at least one aforementioned QTL chain other All combinations of label.

In in terms of some embodiments, the palmitic acid content of sunflower plants can use any conjunction known in the art Suitable means or method are quantified.

II. it abridges

AFLP amplified fragment length polymorphism

ASH allele specific hybridization

CCD charge coupled device

EST EST

FAME fatty acid methyl ester

FID flame ionization detector

GC gas-chromatography

LCR ligase chain reaction

LG linkage group

LNA locked nucleic acid

LOD logarithm probability (being the truth of a matter with 10)

MAS marker assisted selection

Amplification of the NASBA based on nucleic acid sequence

NIR near-infrared (spectrum)

NMR nuclear magnetic resonance (spectrum)

ORF open reading frame

PCR polymerase chain reaction

PNA peptide nucleic acid

QTL quantitative trait locus

RAPD randomly amplified polymorphic DNA

RFLP restriction enzyme fragment length polymorphism

RT-PCR reverse transcriptase-PCR

SNP single nucleotide polymorphism

SSCP single-strand conformation polymorphism

SSR simple sequence repeats

III. term

If this specification includes used in claim, unless clearly defined otherwise, otherwise singular and odd number shape The term of formula, such as " one ", "one" and "the" include multiple indicants.Thus, for example, to " plant ", " plant " or " one The instruction of kind plant " further includes multiple plants.Moreover, based on context, the use of term " plant " also refers to the plant in heredity Upper similar or identical offspring.Similarly, term " nucleic acid " can refer to many copies of nucleic acid molecules.Similarly, term " is visited Needle " can refer to many similar or identical probe molecules.

Digital scope includes the number for defining the range, and including each integer and non-integer point in the confining spectrum Number.Unless otherwise defined, all technical and scientific terms used herein is with general with those of ordinary skill in the art institute All over the identical meaning understood.

In order to facilitate the various embodiments that review describes in the disclosure, the description below of specific term is provided:

Separation: " separation " biological components (such as nucleic acid or protein) are thin with the naturally occurring organism of the component Other biological components (i.e. other chromosomes or exchromosomal DNA and RNA and protein) in born of the same parents are substantially separate, separation Generate or purifying, while influence the component chemistry or changes of function (such as nucleic acid can by fracture connect the nucleic acid It is separated with the chemical bond of remaining DNA in chromosome)." separation " nucleic acid molecules and protein include pure by standard The nucleic acid molecules and protein of change method purifying.The term further includes by recombinantly expressing the nucleic acid of preparation point in host cell Son and protein and chemically synthesized nucleic acid molecules, protein and peptide.

Target group: as it is used herein, term " target group " can refer to plant population's (example of the assignment of genes gene mapping Such as sunflower plants group).Target group usually obtains from the crossing controlled of parent genotype, such as can be by two inbred strais It provides.About Juvenile stage, for development and location group Mating design (mating design) and label used type Decision, depending on gene to be positioned, the availability of label and molecular linkage map.Plant parent in target group should be in core Enough variations are all had to interested character on acid sequence and phenotypic level.The variation of parental nucleic acid sequence is for positioning Recombination event is tracked in the plant of group.The availability of the polymorphism mark of offer information depends on the variation of nucleic acid sequence Amount.Therefore it provides the label of information will not be accredited in the specific cross of parent genotype, the label of even now can be deposited ?.

" genetic map " is that the heredity between the locus (or linkage group) in given species on one or more chromosomes connects The description of lock relationship can be determined by the analysis to target group.In some instances, genetic map can with chart or Form is described.Term " assignment of genes gene mapping " can refer to by using the isolated target group of genetic marker, label and recombination frequency The standard genetic principle of rate defines the process of locus linkage relationship." genetic map position " refers to the position (phase on genetic map For surrounding's genetic marker in identical linkage group or chromosome), it is can be found that on the position in given species A certain specific markers.In contrast, " physical map of genome " refer between given species internal labeling absolute distance (for example, With base-pair or separation and overlapping consecutive gene fragment measurement).The physical map of genome not necessarily reflects on physical map not With the practical recombination frequency observed in the test hybridization of a species between point.

Hybridization；As it is used herein, term " hybridization " or " hybridization " refer to via the Gamete Fusion of pollination to generate Offspring (such as cell, seed and plant).This term includes sexual hybridization (i.e. a plant is pollinated by another) and selfing (i.e. self-pollination, for example, using pollen and ovule from identical plant).

Backcrossing: Backcrossing methods can be used for for nucleic acid sequence being introduced into plant.Baclccrossing techniques have been widely used number 10 years, for new character to be introduced into plant.N.Jensen,Ed.Plant Breeding Methodology,John Wiley&Sons,Inc.,1988.In typical backcrossing scheme, original kind interested (recurrent parent) and carry wait turn The second kind (nonrecurrent parent) of the gene of interest of shifting hybridizes.Then, the resulting offspring of the hybridization is close with circulation again This hybridization, and the process is repeated until obtaining following plant, wherein other than the metastatic gene from nonrecurrent parent, The most expectation morphology of circulation plant and physiologic character are also restored in transformation plant.

(gene) penetrates into: as it is used herein, term " (gene) penetrates into " refers to the equipotential base at some locus Because being transmitted in some genetic background.In some embodiments, the gene transgression of specific allelic form can at locus The allelic form to be transmitted at least one offspring by the sexual hybridization between the two of same species parent Occur, wherein at least one parent has the specific allelic form in its genome.Contain the specific allele The offspring of form can be returned repeatedly with the strain with desired genetic background.Can choose has the specific allelic form Backcross progeny, thus generate a new kind, wherein the specific allelic form has been fastened to the genetic background In.In some embodiments, the gene transgression of specific allelic form can pass through the weight between two donor gene groups (for example, in protoplast of fusion) occurs for group, and wherein at least one donor gene group has this specific in its genome Allelic form.Gene transgression can be related to the transmitting of specific allelic form, which can be, such as But it is not limited only to, the selected allelic form of marker allele；QTL；And/or transgenosis.

Germplasm: as it is used herein, term " germplasm " refers to bion, flora (plant lines, kind and family Race), derived from plant or flora clone it is all or from source genetic stocks.Germplasm can be organism or thin A part of born of the same parents or it can be (such as the separation) separated with organism or cell.Generally, germplasm, which provides, has The genetic stocks of specific molecular composition, is the basis of plant genetic quality.As it is used herein, " germplasm " refers to specific plant The cell of object；Seed；The tissue (for example, tissue that new plant can be grown) of specified plant；With the non-seed portion of specified plant Divide (such as leaf, stem, pollen and cell).

As it is used herein, term " germplasm " is synonymous with " genetic stocks ", it can be used for referring to the seed that can breed plant (or other plant material)." Germplasm Bank " can refer to the difference that can be cultivated known cultivar and can produce new cultivar The organized set of seed or other genetic stocks (wherein every kind of genotype is uniquely identified).In embodiments, exist The germplasm utilized in method as described herein or plant is from Sunflower Lines or kind.In specific example, germplasm It is the seed of Sunflower Lines or cultivar.In specific example, germplasm is the nucleic acid sample from Sunflower Lines or kind Product.

Gene: as it is used herein, term " gene " (or " genetic elements ") can refer to importance functionally Heritable genomic dna sequence.Term " gene " can also be used to refer to, such as but be not limited only to, by heritable genome The cDNA and/or mRNA of DNA sequence encoding.

Genotype: as it is used herein, term " genotype " refers to individual (or groups of individuals) in one or more specific bases Because of the genetic constitution (genetic constitution) at seat.The genotype of individual or groups of individuals is by individual in one or more It defines and describes from the allelic form that its amphilepsis obtains at locus.Term genotype is also used to refer to individual in list Genetic constitution in a locus, multiple locus or its genome at full gene seat." haplotype " is individual in multiple something lost Pass the genotype at locus.In some instances, genetic loci described in haplotype can be physically and hereditarily Chain, i.e., locus can be located on same chromosome segment.

Quantitative trait locus: specific chromosomal loci (or section) can be positioned (map) in the base of organism It is associated with specific quantity phenotype because in group.These locus are referred to as quantitative trait locus or QTL.As made herein , term " quantitative trait locus " (QTL) can refer to the basis for being accredited as that certain quantitative character or phenotype may be constituted DNA sequence dna (such as gene, non-coding sequence, and/or intergenic sequence) DNA section (stretches), the quantitative character or Phenotype is variable in degree, and can be attributed to two or more DNA sequence dnas (such as gene, non-coding sequence, and/or Intergenic sequence) or its expression product and its environment between interaction.Therefore, term " quantitative trait locus " includes tool There is the polymorphic genetic locus of at least two allele, at least two allele is under at least one genetic background (such as at least one breeding population or offspring) can differentially influence phenotypic character expression.It in practice, can be to QTL Molecular Identification is carried out to help to position the base containing the sequence for participating in defined quantitative character (such as reduced palmitic acid content) Because of a group region.

As it is used herein, term " section QTL " can refer to and constitute the DNA of the gene linkage on the QTL character basis Extend.The section QTL is in general, but be not necessarily greater to QTL itself.It the section QTL can be containing corresponding to the QTL's 5 ' and/or 3 ' DNA sections.

It has been developed for a variety of for identifying and analyzing the experimental paradigm of QTL.See such as Jansen (1996) Trends Plant Sci.1:89.It is largely based on bi-parental mating (bi- about the open report positioned of QTL in crop species Parental cross) use (Lynch and Walsh (1997) Genetics and Analysis of Quantitative Traits,Sinauer Associates,Sunderland).Typically, these normal forms include making a pair Or multipair parents, these parents are to can be, for example, the single pairing from two inbreeding strains, or different inbreeding Multiple relationships of strain or strain or without relationship parent, each of which shows different characteristic about phenotypic character interested.It is logical Often, this experimental program is related to from two different inbred strais (for example, these inbred strais are selected to make phenotype between each strain With molecular labeling difference maximize) single hybridization in obtain 100-300 separation offspring.By parent and separation offspring for more A marked locus carries out Genotyping, and assesses one to several quantitative inheritance characters (such as disease resistance).Then, according to The significant correlation of statistics in offspring between genotype numerical value and phenotypic variability is separated as QTL.This experimental program Strong point from inbreeding use, because of resulting F₁Whole chain phase (linkage the phase) (equipotential having the same of parent Juncture of the gene in p generation).Therefore, in F₁After plant selfing, all isolated F₂Offspring can provide information, Linkage disequilibrium (linkage disequilibrium) is maximized, and chain phase is known, QTL equipotential base there are two The frequency of cause, and (in addition to backcross progeny), each QTL allele is 0.5.

It is largely used to determine and marks whether that with the statistical method of QTL (or with another label) genetic linkage be this field skill Known to art personnel, including, such as but be not limited only to, standard linear model such as ANOVA or regression plot (Haley and Knott(1992)Heredity 69:315)；And likelihood method, such as EM algorithm (expectation- Maximization algorithm) (for example, Lander and Botstein (1989) Genetics 121:185-99； Jansen(1992)Theor.Appl.Genet.85:252-60；Jansen(1993)Biometrics 49:227-31； Jansen(1994)“Mapping of quantitative trait loci by using genetic markers:an Overview of biometrical models, " see J.W.van Ooijen and J.Jansen (eds.), Biometrics In Plant breeding:applications of molecular markers, the 116-124 pages, CPRO-DLO Netherlands；Jansen(1996)Genetics 142:305-11；With Jansen and Stam (1994) Genetics 136:1447-55)。

Example statistical method includes single point marker analysis；Deciding field (Lander and Botstein (1989) Genetics 121:185)；Composite interval mapping (composite interval mapping)；Penalized regression analysis (penalized regression analysis)；Complicated pedigree analysis (complex pedigree analysis)；MCMC Analysis；MQM analyzes (Jansen (1994) Genetics 138:871)；HAPLO-IM+ analysis, HAPLO-MQM analysis, and HAPLO-MQM+ analysis；Bayesian MCMC；Ridge regression (ridge regression)；Blood relationship homogeneity analysis (identity-by-descent analysis)；It is returned with Haseman-Elston, either one or two of they are adapted to this hair The context of bright specific embodiment.It can be used for identifying in a particular embodiment and unite with the substitution of the complicated breeding population of Mapping of QTL Meter method discloses in No.WO0149104A2 in United States Patent (USP) 6,399,855 and pct international patent to be described.All these sides Method requires largely to calculate, and usually implements under the auxiliary of computer based system and specific software.Suitable system Meter is learned software package and can be obtained from a variety of public and commercial resource, and is known to the skilled in the art.

Label: although the specific dna sequence of coding protein be generally between species it is very conservative, DNA its Its region (such as noncoding DNA and introne) is intended to develop and accumulate polymorphism, therefore between the individual of same species It can be different.Genome mutation can be any source, for example, variability can be due to DNA insertion, missing, duplication, Repetition DNA element, point mutation, recombination event, and can transposable element sequence presence.These regions may contain useful molecule Genetic marker.Generally, the polymorphism character (including polymorphic nucleic acid) of any difference heredity separated between offspring is Potential label.

As it is used herein, term " label " and " molecular labeling " refer to the product of one section of nucleotide sequence or its coding (such as protein) is used as reference point when identifying linked gene seat.Therefore, label can refer to have spy for identifying Determine the gene or nucleotide sequence of the plant of allele.Label can be described as the variation at given locus.Heredity mark Note can be short dna sequence, such as single base to the sequence of change (single nucleotide polymorphism, or " SNP ") surrounding or one Long sequence, such as microsatellite/simple sequence repeats (" SSR ")." marker allele " or " marker allele form " refers to and deposits It is the version of the label in particular individual.As it is used herein, term " label " can refer to clone's piece of chromosomal DNA Section, can also or alternatively refer to the DNA molecular complementary with chromosomal DNA cloned sequence.The term also refers to and genomic marker The nucleic acid sequence of sequence complementation, such as nucleic acid primer and probe.

Label can be described as, for example, being located at the polymorphic genetic element of the particular locations of organism genetic map.It loses Blit spectrum can be the graphical representation of genome (or a part of genome, such as individual chromosome), wherein the chromosome upper bound The distance between mark (landmark) is measured with the recombination frequency between boundary mark.Hereditary boundary mark can be various known polymorphic Property label any one, such as but be not limited only to: simple sequence repeat (SSR) label；Restrictive fragment length polymerphism (RFLP) Label；It is marked with single nucleotide polymorphism (SNP).As an example, SSR marker can come from genomic nucleic acids or be expressed Nucleic acid (such as EST (EST)).

Others label includes, such as but is not limited only to, ESTs；Amplified fragment length polymorphism (AFLPs) (Vos et al.(1995)Nucl.Acids Res.23:4407；Becker et al.(1995)Mol.Gen.Genet.249:65； Meksem et al.(1995)Mol.Gen.Genet.249:74)；Randomly amplified polymorphic DNA (RAPD) and isoenzyme mark. Isoenzyme mark may be used as genetic marker, for example, for tracking the isodynamic enzyme or other classes chain with specific first label The label of type.Isodynamic enzyme is the enzyme of diversified forms, they on amino acid sequence (therefore in its nucleic acid sequence encoding) each other not Together.Some isodynamic enzymes are the polymer enzymes containing slightly different subunit.Other isodynamic enzymes can be polymer or monomer, be from It is cut down on preemzyme (pro-enzyme), but the site cut in precursor enzyme amino acid sequence is different.Isodynamic enzyme can To characterize and analyze in protein level or in nucleic acid level.Therefore, any method based on nucleic acid as described herein is equal It can be used to analyze isoenzyme mark in particular instances.

" genetic marker " includes the allele in group with polymorphism, and wherein these allele can pass through one Kind or various analysis (such as rflp analysis, aflp analysis, isoenzyme mark analysis, snp analysis, and ssr analysis) are subject to Monitoring and differentiation.Term " genetic marker " can also refer to genetic loci (" marked locus "), can connect in identification heredity It is used as reference point when locking locus (such as QTL).This label may also be referred to as " QTL label ".

The essence of said physical boundary mark (and method for detecting them) is different, but all these labels can root According between polynucleotides length and/or sequence (and between multiple allele of any one specific markers) physically It is distinguish.Have been established for a variety of methods for detection molecules label and identification marking allele.Those skilled in the art It has been known that there is diversified schemes can be used for detecting this variability by member, and these schemes are often and are directed to designed detection Polymorphism type specificity.These schemes include, such as but are not limited only to, PCR amplification；Single-strand conformation polymorphism (SSCP), Such as pass through electrophoresis；With from maintain sequence replicating (self-sustained sequence replication) (3SR) (see Chan and Fox(1999)Reviews in Medical Microbiology 10:185-96)。

From the viewpoint of plant breeder, the mainspring for developing molecular marking technique is that breeding effect is improved by MAS Rate.Prove to be to select in plant population with the molecular labeling allele (such as QTL) of desired phenotypic character linkage disequilibrium Desired character provides useful tool.The key component for executing MAS method is the height for creating molecular labeling in plant germplasm Density (informative) genetic map；At least one QTL is detected according to the correlation statistically between label and phenotypic variability； A series of particularly useful marker alleles are defined according to qtl analysis result；Work as with using and/or being extrapolated to these information In the breeding germplasm of preceding series, determined so as to make the selection based on label.

Hereditary variability, such as determined in target group, it can be in the distinct group of same species (such as sunflower) It is observed between body.Although being likely to occur variability between the Liang Ge group of same species in genetic map, for identification and/ Or selection includes and the plant of plant and/or germplasm and counter-selection comprising bad character for marking chain character and/or kind The purpose of matter, genetic map and mark information from group are still useful generally between multiple groups.

Two kinds of label is SSR marker and SNP marker used in specific MAS method described herein.SSR marker packet Include any molecular heterogeneity type for leading to nucleic acid sequence length variation.The example of SSR marker is that short dna is (for up to hundreds of A base-pair) section, it is made of the multiple tandem sequence repeats of two or three base-pair sequences.Due to replicating fidelity (replication fidelity) poor (such as due to polymerase slippage), these repetitive sequences cause the height of different length more State property region of DNA domain.SSR seemingly random dispersion in genome, two sides are usually conserved region.SSR marker can be from RNA sequence (form in cDNA, part cDNA or EST) and Genomic material.

The heterogeneity of SSR marker makes them be very suitable as molecular genetic marker.For example, SSR genome mutation It is heritable, and is multiple alleles, codominance and repeatable detection.The finer detection skill based on amplification The nucleotide sequence heterogeneity of art (such as technology of based on PCR) being extended between test sample provides diversified spirit Quick method.Probe (such as nucleic acid primer) can be designed to hybridize with the conservative region of the two sides SSR, and these spies can be used The variable area SSR of needle amplification.The different size of amplicon generated from the area SSR has characteristic and reproducible size. Two homologues of some individual from plant population, or the different size of SSR amplification observed from Different Individual Sub-definite SSR marker allele.The SSR marker equipotential that can produce different size of PCR product simply by the presence of at least two Gene, so that it may use the SSR as label.

Chain (no) balance: as it is used herein, term " linkage equilibrium " refers to the case where two label independent separates； I.e. label is randomly assigned (sort) in offspring.The label of display linkage equilibrium be considered as it is not chain (no matter they whether On same chromosome).As it is used herein, term " linkage disequilibrium " refers to two labels in nonrandom mode point From the case where；The recombination frequency marked is less than 50% (therefore according to definition, distance is less than 50cM in same linkage group).? In some embodiments, show that the label of linkage disequilibrium is considered chain.

Chain, close linkage and pole close linkage: as it is used herein, chain between gene and label can refer to such as Under phenomenon, wherein the gene on chromosome or label show the measurable probability for passing to follow-on individual together.Cause This, a label can be measured and/or be indicated with recombination frequency with another label or the chain of gene.Two genes or mark Note is each other closer to this probability is closer to " 1 ".Therefore, term " chain " can refer to one or more genes or label The probability transmitted together with some gene is greater than 0.5, and (0.5 is according to when label/gene is located in different chromosomes free group It closes and expected value).When facilitating certain phenotype in individual there are some gene, can be claimed with the label of the gene linkage Make chain with the phenotype.Therefore, term " chain " can be between the relationship between digit synbol and gene, or label and phenotype Relationship.

Relative Hereditary distance (by exchange frequency determine, and with centimorgan (cM) measurement) generally with two linked markers or base Physical distance because between on chromosome is proportional.One centimorgan is defined as 1% recombination frequency of display (that is, between two labels Occur primary to exchange event in every 100 cell divisions) the distance between two genetic markers.Generally, a label With another label or gene closer to (no matter they the distance between be to be measured with genetic distance or surveyed with physical distance Amount), they are closelyr chain.Because chromosome distance is approximate proportional to the recombination event frequency between character, deposit In an approximate physical distance associated with recombination frequency.For example, in sunflower, on average, 1cM and about 400kb Association.

Therefore, term " chain " can refer to physical location between on same sunflower chromosome herein One or more genes or label within about 4.0Mb (i.e. about 10cM).Therefore, two " chain " genes or label It can be at a distance of 4.1Mb；About 4.0Mb；About 3.0Mb；About 2.5Mb；2.1Mb；2.00Mb；About 1.95Mb；About 1.90Mb；About 1.85Mb；About 1.80Mb；About 1.75Mb；About 1.70Mb；About 1.65Mb；About 1.60Mb；About 1.55Mb；About 1.50Mb；About 1.45Mb；About 1.40Mb；About 1.35Mb；About 1.30Mb；About 1.25Mb；About 1.20Mb；About 1.15Mb；About 1.10Mb；About 1.05Mb；About 1.00Mb；About 0.95Mb；About 0.90Mb；About 0.85Mb；About 0.80Mb；About 0.75Mb；About 0.70Mb；About 0.65Mb；About 0.60Mb；About 0.55Mb；About 0.50Mb；About 0.45Mb；About 0.40Mb；About 0.35Mb；About 0.30Mb；About 0.25Mb；About 0.20Mb；About 0.15Mb；About 0.10Mb；About 0.05Mb；About 0.025Mb；About 0.01Mb.

As it is used herein, term " close linkage " can refer on same chromosome each other at a distance of about One or more genes or label within 2.0Mb.Therefore, two " close linkage " genes or label can be at a distance of 2.1Mb； About 1.75Mb；About 1.5Mb；About 1.0Mb；About 0.9Mb；About 0.8Mb；About 0.7Mb；About 0.6Mb；About 0.55Mb；About 0.5Mb；About 0.45Mb；About 0.4Mb；About 0.35Mb；About 0.3Mb；About 0.25Mb；About 0.2Mb；About 0.15Mb；About 0.1Mb；About 0.05Mb.

As it is used herein, term " pole close linkage " can refer on same chromosome each other at a distance of big One or more genes or label within about 500kb.Therefore, two " pole close linkage " genes or label can be apart 600kb；About 450kb；About 400kb；About 350kb；About 300kb；About 250kb；About 200kb；About 175kb； About 150kb；About 125kb；About 120kb；About 115kb；About 110kb；About 105kb；100kb；About 95kb；Greatly About 90kb；About 85kb；About 80kb；About 75kb；About 70kb；About 65kb；About 60kb；About 55kb；About 50kb；About 45kb；About 40kb；About 35kb；About 30kb；About 25kb；About 20kb；About 15kb；About 10kb； About 5kb；About 1kb.

One specific markers is closer at a distance from the gene for the polypeptide that coding contributes to some particular phenotype (either with something lost Pass distance or measured with physical distance), the specific markers and the phenotype get over close linkage.In view of above-mentioned, it will be appreciated that, with Specific gene or the chain label of phenotype include the label with the gene or phenotype close linkage, and the mark of pole close linkage therewith Note.In some embodiments, specific markers are facilitated closer at a distance from the gene of the polypeptide of low palmitic acid content phenotype with coding (either still being measured with physical distance with genetic distance), the specific markers and low palmitic acid content phenotype get over close linkage. Therefore, in sunflower with low palmitic acid content phenotype is chain, the genetic marker of close linkage and pole close linkage can be used in MAS journey In sequence, contain reduced palmitic acid content for identifying (with parental breed and/or at least one specific environment variety protection) Sunflower Varieties, identification contain reduced palmitic acid content single sunflower plants, and by this character breeding to it is other to In day certain herbaceous plants with big flowers kind, to reduce palmitic acid content.

In some embodiments, the linkage relationship between molecular labeling and phenotype can use " probability " or " adjustment probability " It indicates.In the context, probability value is that " phenotype and the present or absent specific combination of specific markers allelic form are It is random " statistics likelihood.Therefore, probability score is lower, and phenotype separates jointly with specific markers allelic form Likelihood is bigger.In some embodiments, probability score can use " significant " or " not significant " description.In specific example, The probability score of random combine is that 0.05 (p=0.05 (5% probability)) is considered as " significant " instruction separated jointly.However, In other embodiments, significant probability can be any probability less than 50% (p=0.5).For example, significant probability can be less than 0.25；Less than 0.20；Less than 0.15；Or less than 0.1.

In some embodiments, the label chain with low palmitic acid content phenotype can connect from sunflower shown in Fig. 4 It is selected in the QTL label of lock group 5.In some embodiments, the label chain with low palmitic acid content phenotype can from Fig. 4 Shown in QTL label selected in the label within the about 10cM.Therefore, the label chain with low palmitic acid content phenotype can It can mark with QTL shown in Fig. 4 at a distance of such as 10cM；9cM；8cM；7cM；6cM；5cM；4cM；3cM；2cM；1cM；0.75cM； 0.5cM；0.25cM；Or less distance.

Molecular labeling can be advantageously used in plant breeder, pass through identification and desired phenotype (such as low palmitic acid content) It shows the statistically significant marker allele for isolating probability, shows as linkage disequilibrium, to identify desired individual. By identifying thus the molecular labeling isolated with quantitative character or molecular labeling group, breeder identify a QTL.Pass through identification With selection marker allele (or expectation allele from multiple labels) associated with desired phenotype, plant breeder It can be by selecting suitable molecular labeling allele to be rapidly selected phenotype (i.e. MAS).The molecule being placed on genetic map Label is more, and the figure is for instructing the potentially useful of MAS bigger.

Mark group: can be used for identifying comprising character interested as it is used herein, one " group " label or probe refer to The label of individual or the specific collection of probe.In some embodiments, it can be used with the chain label of low palmitinic acid phenotype for one group In the sunflower plants that identification includes low palmitic acid content.(or these marks are used corresponding to the data of mark group or probe groups The data that note or probe obtain) it can store in electronic media.Although each of mark group label is used equally for character Identification, but the individual labels selected in the subgroup from mark group and comprising some but not all label are also effectively used for reflecting It surely include the individual of character interested.

Allele: as it is used herein, term " allele " refers to two present in some particular locus One in a or multiple and different nucleotide sequences.For example, the first allele can appear on a chromosome, and second Allele can appear on the second homologue；For example, appearing in the different chromosomes of heterozygote individual, Huo Zhequn Between the different homozygotes or heterozygote individual of body.In some embodiments, the specific allele at particular locus can With chain with the desired phenotype of agronomy (such as low palmitic acid content).In some embodiments, specific etc. at locus Position gene allow identify do not include agronomy expectation phenotype plant (such as high palmitic acid content plant), so as to by those Plant removes from the procedure of breeding or plantation.Marker allele can separate together with beneficial phenotype, thus provide mirror It surely include the income of the phenotype plant." allelic form of chromosome segment " can refer to comprising such marker allele The chromosome segment of nucleotide sequence, the marker allele nucleotide sequence contribute to particular phenotype or connect with particular phenotype Lock, the particular phenotype physical positioning is at one or more genetic locis on chromosome segment.

" gene frequency " can refer to that allele is located at some locus in plant, in strain or in strain group The frequency (being indicated with ratio or percentage) at place.Therefore, for allele " A ", genotype is " AA, " " Aa, " or " aa " The gene frequency of dliploid individuality is respectively 1.0,0.5 or 0.0.Gene frequency in strain can be by that will come from The gene frequency of the individual sample of the strain, which is averaged, to be estimated.Similarly, the intragroup allele frequency of strain Rate can be calculated by the way that the gene frequency for constituting the strain of the group to be averaged.There is Finite Number for one The group of mesh individual or strain, gene frequency can use the individual containing the allele or strain (or any other rule Fixed grouping) counting indicate.

When marker allele and the linkage of characters, and the presence instruction of the marker allele includes the plant of the allele When will appear the anticipant character or character form in object, marker allele is related to character " just ".When label and the linkage of characters, And the presence instruction of the marker allele is comprising being not in the anticipant character or character form in the plant of the allele When, marker allele is related to character " negative ".

" homozygosis " individual only has a form of allele at given locus, and (such as diplont is at two With the copy of identical allelic form at the particular locus of each of homologue).If deposited at locus Be more than a kind of allelic form, then individual be " heterozygosis " (such as dliploid individuality at locus have the first equipotential One copy of gene forms and a copy of the second allelic form).Term " homozygosity " refers to that the member of group exists Genotype (i.e. identical gene frequency) having the same at one or more specific gene of interest seats.On the contrary, term is " miscellaneous Conjunction property " refers to that the individual in group has different genotype at one or more specific gene of interest seats.

Any technology that can be used for characterizing the nucleotide sequence at some locus is used equally for identification marking allele. For marker allele detection method include, such as but be not limited only to, method for identifying molecules (for example, amplification and label expand Increase the detection of son).For example, the allelic form of SSR marker or SNP marker can be examined by the technology based on amplification It surveys.In the detection method typically based on amplification, a part of marked locus or marked locus is amplified (using for example PCR, LCR and transcription are amplification template with the nucleic acid separated from interested sunflower plants), and detect resulting through expanding Label amplicon.In some embodiments, template of the plant RNA as amplified reaction can be used.In some embodiment party In case, template of the plant genome DNA as amplified reaction can be used.In some instances, QTL label is SNP marker, institute The allele of detection is SNP marker allele, and detection method is allele specific hybridization (ASH).Some In example, QTL label is SSR marker, and allele detected is SSR marker allele.

ASH technology is the stabilizing annealing of the single-stranded target nucleic acid based on short single strand oligonucleotide probes and complete complementary.Detection It can be realized by detecting the isotope being attached on probe or non-isotopic labels.For every kind of polymorphism, two or more A different ASH probe can be designed to DNA sequence dna having the same, in addition at polymorphic site.Each probe can be with One allelic sequences is completely homologous, so that the range of probe can distinguish all known optional allelic sequences. When each probe suitable probe design and hybridization conditions under hybridize with target DNA when, single base between probe and target DNA is wrong With can prevent to hybridize.In this way, only one may be selected probe can be homozygous target sample for allele Hybridization.It is that heterozygosis or heterogeneous sample can then hybridize with two optional probes simultaneously for two allele.

ASH label may be used as dominant marker, wherein not hybridizing from the hybridization of only one probe or and determining only one etc. The existence or non-existence of position gene.Selectable allele can infer from the missing of hybridization.In instances, ASH probe and Target molecule can be RNA or DNA molecular；Target molecule may include the nucleosides of any length except the sequence with probes complementary Acid；Probe can be designed to hybridize with any chain of DNA target；And the size of probe can be different, to meet different hybridization items The requirement of part.

The variable sequence of amplification refers to such extension increasing sequence of Plant Genome, it shows between the member of same species Show the nucleotide residue variability of height.All organisms all have variable genome sequence, and each organism (other than clone) all has a different set of variable sequence.Once identifying the presence of particular variable sequence, can be used for pre- Survey phenotypic character.In some embodiments, the DNA from plant can be used as template, with the primer for being located at DNA variable sequence two sides It is expanded.Then the variable sequence, which can be amplified, to be sequenced.

It can also be alternatively for identification genetic marker from maintenance sequence replicating.Refer to use in base from maintenance sequence replicating The nucleic acid amplification method of the target nucleic acid sequence exponentially replicated in vitro under conditions of isothermal in sheet is reversed using three kinds of participations Record the enzymatic activity of virus replication: reverse transcriptase；RNA enzyme H；The RNA polymerase relied on DNA.Guatelli et al.(1990) Proc.Natl.Acad.Sci.USA87:1874.By the rna replicon plan for simulating retrovirus using cDNA intermediate product Slightly, which accumulates cDNA and the RNA copy of initial target.

The data for representing detected marker allele can be transmitted (for example, electron-transport；And by infrared, wireless Or optics conveying) computer or computer-readable medium are given, for analyzing and storing.

For example, amplimer or amplimer are to the genomic nucleic acids that can be separated with from the first sunflower plants or germplasm Mixing, wherein primer or primer pair is complementary at least part of marked locus or partial complementarity, and primer or primer pair Sunflower genomic nucleic acids are able to use as template, DNA polymerization is caused by archaeal dna polymerase.In DNA polymerization reaction, primer Or primer pair (such as the primer pair provided in table 6) is extended using archaeal dna polymerase and templet gene group nucleic acid, generates at least one Amplicon.

" positional cloning " refers to a kind of special Cloning processes, wherein according to the genome degree of approach of target nucleic acid and label (genomic proximity) is identified and isolated from target nucleic acid.For example, genomic nucleic acids clone may include closer to each other two Or all or part of multiple chromosomal regions.If label can be used in identifying genomic nucleic acids clone from genomic library, Standard method, such as subclone and/or sequencing can be used, identification and/or separation are located at the subsequence of the clone near label.

Locus: as it is used herein, term " locus " refers on genome corresponding to can measure feature (such as property Shape) or polymorphism position.SNP locus with the probe for the DNA hybridization for including in locus by that can define.

Marker-assisted breeding: as it is used herein, term " marker-assisted breeding " can refer to directly using MAS to one Or the method that multiple characters (such as low palmitic acid content) carry out breeding.In current practice, plant breeder attempts to identify Easily the detect and chain character of agronomy anticipant character, such as pattern, kind skin appearance or isoenzyme variant.Plant breeder The Agronomic character is then tracked in isolated breeding population by tracking the separation of the easy detection character.However, these connect Only have only a few to can be used for plant breeding in lock relationship.

Marker-assisted breeding provides the method for high time efficiency and cost effective for improvement plant variety.Using label Some examples of assistant breeding are related to using isoenzyme mark.See such as Tanksley and Orton, eds. (1983) Isozymes in Plant Breeding and Genetics,Amsterdam:Elsevier.One example be in tomato The isoenzyme mark of the gene-correlation of anti-nematode evil.The resistance is located at No. 6 chromosomes of tomato by naming the gene control for Mi On, with a kind of Aps1 (Acid Phosphatase Isozymes) pole close linkage.It is mentioned using Aps1 isoenzyme mark indirect selections Mi gene For advantage be: the separation of group can standard electrophoretic techniques clearly determined；Can give in seedling tissue should Isoenzyme mark marking, without maintaining plant to maturation；And the codominance of the isoenzyme mark allows to distinguish homozygote And heterozygote.The Rick (1983) in Tanksley and Orton is seen, with above.

Probe: in some embodiments, mark the presence in plant that can be detected by using nucleic acid probe. Probe can be DNA molecular or RNA molecule.Rna probe can be synthesized by methods known in the art, such as use DNA points Subtemplate.Probe may include all or part of of labeled nucleotide sequence, and the additional neighbour from Plant Genome Connect nucleotide sequence.This is herein referred to as " adjacent probe (contiguous probe) ".Additional adjoining nucleotides sequence Column are referred to as " upstream " or " downstream " of former probe, depend on according to conventional understanding, should the adjoining nucleotide from plant chromosome Sequence is located at the 5 ' sides or 3 ' sides of former label.It will be appreciated by those skilled in the art that obtain additional adjacent nucleotide sequence with Include that process in label can repeat (only by the length limitation of chromosome) secondaryly with virtually limitless, reflect whereby along chromosome Label outside quota.

Sequence oligonucleotide probe can be synthetically prepared or be prepared by clone.Suitable cloning vector is ability Field technique personnel are well-known.Oligonucleotide probe can be labeled or not mark.There are many for marker nucleic acid molecule Technology, including for example but be not limited only to: nick translation radioactive label (radiolabeling by nick translation)；Random priming；Deoxynucleotidyl transferase tailing (tailing with terminal Deoxytransferase), etc., nucleotide such as radioactivity used in³²P label.Other available marker packets It includes, such as but be not limited only to: fluorogen (such as FAM and VIC)；Enzyme；Zymolyte；Enzyme co-factor；Enzyme inhibitor；Deng.Alternatively, Can be used with report in conjunction with ligand come instead of using being provided by itself or provided together with other reactants detectable letter Number marker, wherein report labeled (such as passing through above-mentioned label) with provide detectable signal (provided by itself or with Other reactants provide together).See such as Leary et al. (1983) Proc.Natl.Acad.Sci.USA80:4045-9.

Probe can contain nucleotide sequence not adjacent with former label；This probe is herein referred to as " non-adjacent Probe (noncontiguous probe) ".The sequence of non-adjacent probe is close enough with former flag sequence in genome, from And make the non-adjacent probe and mutually homogenic or character (such as low palmitic acid content) genetic linkage.For example, in some embodiment party In case, non-adjacent probe is about 10cM at a distance from the label of QTL shown in Fig. 4；9cM；8cM；7cM；6cM；5cM；4cM；3cM； 2cM；1cM；0.75cM；0.5cM；0.25cM；Or it is smaller.

Probe can be the definite copy for the label to be detected.Probe be also possible to comprising following nucleotide sequence or by The nucleic acid molecules that following nucleotide sequence is constituted: basic with the cloned sequence of theme organism (such as sunflower) chromosomal DNA Upper identical nucleotide sequence.As it is used herein, term " substantially the same " can refer to higher than 85% identical nucleotide Sequence.For example, substantially the same nucleotide sequence can be with reference sequences 85.5%；86%；87%；88%；89%； 90%；91%；92%；93%；94%；95%；96%；97%；98%；99% or 99.5% is identical.

Probe can also be can be with the definite copy " specific hybrid " of the label to be detected (" DNA target ") or " special Property it is complementary " nucleic acid molecules.Term " can (with ...) specific hybrid " and " (with ...) specific complementary " refer to sufficiently The complementarity of degree, so that stable and special combination occur between nucleic acid molecules and DNA target.It is capable of the core of specific hybrid Acid molecule need not be complementary with the target sequence 100% that it specific can hybridize therewith.When the complementarity there are abundant degree is to keep away Exempt from nucleic acid molecules and non-target sequences under conditions of desired specific bond, such as under stringent hybridization conditions, occurs non-specific In conjunction with when, nucleic acid molecules can specific hybrid.

Cause the hybridization conditions of specific degrees stringency can be with the essence and hybrid nucleic acid sequence of selected hybridizing method Composition and length and change.Generally, ionic strength (the especially Na of hybridization temperature and hybridization buffer⁺And/or Mg⁺⁺Concentration) The stringency that can determine whether hybridization, although scavenging period also will affect stringency.It is miscellaneous needed for specific degrees stringency about realizing The calculating of friendship condition is known to the skilled in the art, and is discussed in such as following documents: the (eds.) such as Sambrook Molecular Cloning:A Laboratory Manual, the second edition, the 1-3 volumes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, the 9th and 11 chapters；With Hames and Higgins (eds.) Nucleic Acid Hybridization,IRL Press,Oxford,1985.About nucleic acid hybridization more detailed description and Guidance may refer to, such as Tijssen, " Overview of principles of hybridization and the Strategy of nucleic acid probe assays, " referring to Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, I Part, the 2nd chapter, Elsevier, NY, 1993；It is compiled with Ausubel etc., Current Protocols in Molecular Biology, the 2nd chapter, Greene Publishing and Wiley-Interscience, NY, 1995.

As it is used herein, " stringent condition " includes following condition, under this condition, only hybrid molecule and DNA Mispairing between target can just hybridize when being less than 50%." stringent condition " includes more specific horizontal stringency.Therefore, such as this Used in text, " medium stringent " condition refers to following condition, wherein the molecule more than 50% mispairing will not hybridize；" Gao Yan Lattice " condition refers to following condition, wherein the molecule more than 20% mispairing will not hybridize；" high stringent " condition refers to following Condition, wherein the molecule more than 10% mispairing will not hybridize.

Here is representative non-limiting hybridization conditions.

High stringency (detecting the sequence at least 90% sequence identity): miscellaneous in 65 DEG C of 5x SSC buffers It hands over 16 hours；It is cleaned in room temperature 2x SSC buffer 2 times, every time 15 minutes；It is cleaned in 65 DEG C of 0.5x SSC buffers 2 times, every time 20 minutes.

High stringency (detects the sequence at least 80% sequence identity): in 65-70 DEG C of 5x-6x SSC buffer Middle hybridization 16-20 hours；It is cleaned 2 times in room temperature 2x SSC buffer, it is 5-20 minutes each；Delay in 55-70 DEG C of 1x SSC It is cleaned in fliud flushing 2 times, every time 30 minutes.

Medium stringency (detects the sequence at least 50% sequence identity): in -55 DEG C of 6x SSC buffers of room temperature Middle hybridization 16-20 hours；It is cleaned 2 times in -55 DEG C of 2x-3x SSC buffers of room temperature, it is 20-30 minutes each.

About previously discussed all probes, probe may include additional nucleic acid sequence, such as promoter；Transcription letter Number；And/or carrier sequence.Previously discussed any probe is used equally for defining and participates in low palmitic acid content in sunflower The mutually chain additional markers of gene, the label so identified can be of equal value with the example markup named in the disclosure, therefore at this Within the scope of invention.

Sequence identity: as it is used herein, in two nucleic acid or the context of polypeptide sequence, " sequence is same for term One property " or " identity " can refer to when two sequences are aligned in the way of having maximum phase response in specific comparison window When, identical residue in two sequences.

As it is used herein, term " Percent sequence identity " can refer to by comparing two in comparison window most The numerical value that the sequence (such as nucleotide sequence) of good alignment determines, wherein in comparison window, a part of sequence with refer to sequence Column may include addition compared to (it does not include addition or missing) or lack (i.e. vacancy (gap)) to realize the best of two sequences Alignment.The calculating of percentage is the number by determining the position for occurring identical nucleotide or amino acid residue in the two sequences To generate the number of matching position, by the number of matching position divided by the sum of position in comparison window, then by result multiplied by 100, obtain Percent sequence identity.

For aligned sequence so that the method compared is well-known in the art.Various programs and alignment algorithm are for example It is described in following documents: Smith and Waterman (1981) Adv.Appl.Math.2:482；Needleman and Wunsch(1970)J.Mol.Biol.48:443；Pearson and Lipman(1988) Proc.Natl.Acad.Sci.U.S.A.85:2444；Higgins and Sharp(1988)Gene73:237-44；Higgins and Sharp(1989)CABIOS 5:151-3；Corpet et al.(1988)Nucleic Acids Res.16:10881- 90；Huang et al.(1992)Comp.Appl.Biosci.8:155-65；Pearson et al.(1994)Methods Mol.Biol.24:307-31；Tatiana et al.(1999)FEMS Microbiol.Lett.174:247-50.Sequence ratio The detailed description calculated method and homology can be in such as Altschul et al. (1990) J.Mol.Biol.215: It is found in 403-10.

Basic Local Alignment Search Tool (the BLAST of National Center for Biotechnology Information (NCBI)^TM；Altschul et Al. (1990)) it can be obtained from several resources, including National Center for Biotechnology Information (Bethesda, MD) and mutual In networking, for being used together with a variety of sequence analysis programs.On how to use the program to determine the description of sequence identity It can be on the internet in BLAST^TM" help " part obtain.In order to compare nucleic acid sequence, BLAST can be used^TM (Blastn) " Blast 2sequences " function of program, uses the default BLOSUM62 matrix system for being set as default parameter Column.When being estimated with this method, with reference sequences there is the nucleic acid sequence of bigger similitude will show bigger percentage Identity.

Nucleic acid molecules:, can be with as it is used herein, term " nucleic acid molecules " can refer to the multimeric forms of nucleotide It simultaneously include RNA, cDNA, genomic DNA and above-mentioned synthesized form and the sense and antisense chain for mixing polymer.Nucleotide It can refer to the modified forms of ribonucleotide, deoxyribonucleotide or two kinds of any type nucleotide.As used herein , " nucleic acid molecules " are synonymous with " nucleic acid " and " polynucleotides ".The term includes the single-stranded and double-stranded form of DNA.Nucleic acid molecules It may include that the natural generation to be linked together by the nucleotide connecting key naturally occurred and/or non-natural occurs and process are repaired The nucleotide of decorations it is any one or two kinds of.

It is not intrinsic point of particular system (such as germplasm, kind, excellent variety, and/or plant) that " external source " molecule, which refers to, Son refers to its nucleotide sequence and/or genome positioning for polynucleotides, its amino acid sequence is referred to for polypeptide Column and/or cellular localization.In embodiments, external source or heterologous polynucleotide or polypeptide, which can be, is artificially added to biosystem (such as plant cell, plant gene, specified plant species or cultivar and/or plant chromosome), not the particular organisms system It unites intrinsic molecule.Therefore, a nucleic acid being appointed as " external source " can indicate that the nucleic acid occurs except source from natural Source, or can indicate that nucleic acid has non-natural configuration, Genes location and element arrangements.

On the contrary, for example, " natural " or " endogenous " nucleic acid refer to chromosome in addition to normally occurring naturally in the nucleic acid or its On his genetic stocks except the nucleic acid elements of normal presence, the nucleic acid (such as gene) without containing other nucleic acid elements.Endogenous base Because transcript is by the nucleotide sequence coded of native chromosomal locus, rather than artificially it is added to cell.

Term " recombination " refers to material (such as recombinant nucleic acid, recombination, the recombination being changed by human intervention Polynucleotides and/or recombinant polypeptide).For example, the part of recombinant molecule or the arrangement of element can not be natural alignment, and/or The primary sequence of recombinant molecule can be different from its native sequences in certain mode.Material be can change in its natural ring Recombined material is generated or therefrom removed in border or state.If the nucleotide sequence of open reading frame is by from its natural context Middle removal is simultaneously cloned into any kind of artificial nucleic acid (such as carrier), then the open reading frame of nucleic acid is recombination.It produces The scheme and reagent of raw recombinant molecule especially recombinant nucleic acid, are that this field is common and conventional.Herein, term " recombination " It can also refer to the cell comprising recombined material or organism (such as plant and/or plant cell comprising recombinant nucleic acid).One In a little examples, recombinant organisms are transgenic organisms.

As it is used herein, term " introducing " instruction will heterologous or exogenous nucleic acid it is indexable arrive it is intracellular when, be to instigate Nucleic acid is imported into the cell by the methodology that can be obtained with any this field.The term includes nucleic acid introducing method, including example It such as but is not limited only to, transfects；Conversion；And transduction.

As it is used herein, term " carrier " is to refer to a near few nucleic acid fragment to be transferred to intracellular multicore Thuja acid or other molecules.Carrier can optionally include the component/member for mediating carrier to keep and can realizing desired use Part (such as required sequence is replicated, the gene of drug or antibiotic resistance, multiple cloning sites are assigned, and/or make to be cloned gene Promoter/the enhancer element being operatively connected that can be expressed).Carrier can come from, for example, plasmid, bacteriophage or plant Or animal virus." cloning vector ", " shuttle vector " or " subcloning vector " generally comprises the element being operatively connected, easily to change Clone or subcloning steps (for example, the multiple cloning sites for containing multiple restriction endonuclease sites).

As it is used herein, term " expression vector " refers to comprising can easily change coded sequence in specific host organism The carrier for the polynucleotide sequence of expression being operatively connected.For example, bacterial expression vector can easily change coded sequence in bacterium Interior expression.Plant expression vector can easily change expression of the coded sequence in plant cell.Table of easyization in prokaryotes The polynucleotide sequence reached may include, such as but be not limited only to, promoter；Operator；And ribosome bind site.Eukaryon Promoter that biological expression carrier (such as plant expression vector) includes, enhancer, terminator and polyadenylation signal (and its His sequence) generally with difference used in prokaryotic expression carrier.

Single nucleotide polymorphism: as it is used herein, term " single nucleotide polymorphism " (SNP) can refer to when some object There is single nucleosides between the member of kind or between pairing chromosome of some individual on genome (or other share sequences) The mutant dna sequence of sour difference Baoan Member.In group, SNP can be assigned (assigned) lesser allele Frequency is the minimum gene frequency at some locus observed in special group.It is more that here it is mononucleotides In two gene frequencies of state property it is smaller that.It is expected that different groups shows at least small different allele frequency Rate.Specific group can show dramatically different gene frequency.In some embodiments, the mark chain with SCN resistance Note is SNP marker.

SNP can be located in the intergenic region between the coded sequence of gene, the noncoding region or gene of gene.By SNP in the degeneracy of genetic codon, coded sequence not necessarily changes the amino acid sequence of generated protein.Two kinds Form causes the SNP of identical polypeptide sequence to be referred to as " synonymous " (sometimes referred to as silent mutation).If generated different Polypeptide sequence, then they are referred to as " non-synonymous ".Change non-synonymous can be missense or nonsense, wherein missense change Cause to generate different amino acid, and nonsense changes the terminator codon caused ahead of time.SNP pairs not in protein coding region Gene montage, transcription factor combine or the sequence of non-coding RNA remains on and has an impact.SNP is usually diallele, therefore It is easy to measure in plant and animal.Sachidanandam(2001)Nature 409:928-33.

Plant: as it is used herein, term " plant " can refer to entire plant, the cell or tissue training derived from plant Support object and/or any one aforementioned any part.Therefore, term " plant " includes, such as but is not limited only to, entire plant；It plants Object component and/or organ (such as leaf, stem and root)；Plant tissue；Seed；And plant cell.Plant cell can be, such as but It is not limited only to, in plant and/or the cell of plant, the cell separated from plant, and by cultivating the cell separated from plant The cell of acquisition.Therefore, term " sunflower plants " can refer to, such as but be not limited only to, entire sunflower plants；It is multiple to day Certain herbaceous plants with big flowers plant；Sunflower plant cell；Sunflower plants protoplast；Sunflower tissues culture (such as sunflower can be regenerated Plant person)；Sunflower plants callus；Sunflower plants part (such as sunflower seeds, sunflower flower, sunflower cotyledon, Sunflower leaf, sunflower stem, sunflower bud, sunflower root and the sunflower tip of a root)；With in sunflower plants or to day It is complete sunflower plant cell in the part of certain herbaceous plants with big flowers plant.

" genetically modified plants " be at it at least one into the cell containing the plant of exogenous polynucleotide.In instances, external source Polynucleotides by stable integration in the genome of cell, to keep the polynucleotides hereditary in the continuous generation.? In some examples, a part that heterologous polynucleotide can be used as recombinant expression cassettes is incorporated into genome.Herein, art Language " transgenosis " is for referring to the cell that its any genotype has been changed due to the presence of exogenous nucleic acid, cell line, callus group It knits, organize, plant part or plant.Therefore, which includes the transgenosis life for being initially changed and include exogenous polynucleotide Object and cell and those organisms generated by the hybridization or vegetative propagation of initial transgenic organism or cell and thin Born of the same parents.As it is used herein, term " transgenosis " does not include by traditional plant breeding method (for example, only non-transgenic organisms The hybridization of body) or it is (such as random allogamy, non-recombinant virus infection, non-recombinant Bacterial Transformation, non-by naturally-occurring event Recombinate swivel base and spontaneous mutation) and genome (outside the chromosome or chromosome) variation of introducing.

Plant " strain ", " kind " or " strain (being) " are the groups with the bion of identical parent.Plant in strain Object generally has a degree of inbreeding, and is generally homozygous and homogeneous at most of genetic locis." subbreed " can be with Refer to some inbreeding offspring's subgroup from common ancestor, with other similar inbreeding subgroups for multiplying from phase identical forebears in heredity On can distinguish.In some embodiments, " subbreed " can be generated by following means: be used to leisure F₃-F₅The individual that generation is selected The seed inbreeding of sunflower plants, until separation locus remaining in most of or whole locus is that homozygosis is Only.

Business Sunflower Varieties collect single generally by making two genetically crossing controlleds between different parent F₃-F₅For the self-pollination progenies (" put together (bulking) ") of plant.The kind of even now is usually seemingly uniform, still Self-pollination kind from selected plant is final (for example, in F₈Before generation) it will become the mixture of homozygote plant, these are pure Plant is closed in initial selected F₃-F₅It is different for the genotype at any locus of heterozygosis in plant.In embodiment party as described herein In case, by selected F₃-F₅The seed sample of the self-pollination progenies individual of plant carries out Genotyping and produces based on mark The subbreed of note, these subbreed based on one or more the number tag polymorphism on the DNA level at particular locus and each other It is different.Such seed sample can carry out Genotyping directly as seed, or as the plant tissue from seed growth Carry out Genotyping.In some instances, the seed that will there is collaborating genes type at one or more expression of specific marker genes seats It puts together together and generates subbreed, the subbreed is in the one or more bases chain with character interested (such as low palmitic acid content) Because being hereditary homozygous at seat.

" ancestral system " refers to the parent for being used as or being utilized as genetic stocks source (such as developing excellent strain) System." ancestor group " refer to be the ancestors that main body for developing the hereditary variation of excellent strain made contribution group Body." offspring " is the descendants of ancestors, and offspring can be separated by breeding and its ancestors in many generations.For example, excellent strain is it The offspring of ancestors." pedigree " can be used for describing the relationship between offspring and each of which ancestors.Pedigree can cross over a generation or mostly generation, Therefore it can describe mutually to be interposed between 1,2,3,4 relationships for waiting algebra between offspring and its ancestors.

" excellent strain " or " elite plant strain " refers to by breeding and selects superior agronomy performance (frequently by more pollings Select) the superior strain of agronomy.It is available that there are many excellent Sunflower Lines, and is known to the skilled in the art.It is excellent Good group is a group excellent strain or the individual from excellent strain, they can be used for representing given crop species (such as to day Certain herbaceous plants with big flowers) the state-of-art in terms of available agronomy superior genotypes.Similarly, the elite plant of elite germplasm or germplasm System is the superior germplasm of agronomy.Elite germplasm can be obtained from the plant with superior agronomy performance, and can be used in Generate the plant with superior agronomy performance, such as sunflower excellent strain that is existing or developing recently.

Opposite with excellent strain, " external strain " or " external strain " (or " Exotic Germplasm ") refers to available from being not belonging to The strain or germplasm that the sunflower of excellent Sunflower Lines or germplasm strain obtains.In being mixed with for two sunflower plants or germplasm In the context of friendship, the elite germplasm that Exotic Germplasm is hybridized with it is alive to fasten not nearly edge closely.Most commonly, denizen Matter is selected in order to which novel genetic elements (such as interested allelic form) are introduced into the procedure of breeding.

Character or phenotype: term " character " and " phenotype " are used interchangeably herein, and refer to measurable or observable Heritable feature.In some instances, phenotype can be directly by individual gene or genetic loci control (that is, monogenic Shape).In other instances, phenotype can be the result (complex character) of several gene interactions.Therefore, QTL can pass through Single-gene mechanism is acted on by polygenes mechanisms play.In some instances, character or phenotype can be designated " a table Offset " corresponds to the quantitative values measured for the phenotypic character.

Term " molecular phenotype " can refer to the phenotype detected in the level of the group of (one or more) molecule. In some instances, molecular phenotype can only can detect on a molecular scale.The detectable molecule of phenotype can be nucleic acid (such as genomic DNA or RNA)；Protein；And/or metabolin.For example, molecular phenotype can be one or more gene products Expression general picture (profile) (such as the expression general picture of the moment in development of plants, or response environmental condition or stress Express general picture).

Low palmitic acid content: for the purpose of this disclosure, character of special interest is " low palmitic acid content ".Although to The fatty acid composition of day certain herbaceous plants with big flowers plant will receive the influence of environmental factor to a certain extent, but those skilled in the art can manage Solution, palmitic acid content (and other oiliness shapes) are mainly to be determined by heritable inherent cause.Thus, for example, it is specific to The selection of day certain herbaceous plants with big flowers kind can be based at least partially on the certain species in normal field growing conditions (such as without arid, disease With the condition of appropriate soil nutrient) under feature palmitic acid content.In instances, the sunflower with low palmitic acid content is planted Palmitinic acid (16:0) content that object is included can account for about 3% or lower of total oil content in the seed of the plant.One In a little examples, the palmitic acid content that this sunflower plants with low palmitic acid content are included can account for the seed of the plant In total oil content about 2.5% or lower, such as but be not limited only to, palmitic acid content can be 2.6%；2.5%； 2.4%；2.3%；2.2%；2.1%；2.0%；1.9%；1.8%；About 1.7%；It is lower.

In some embodiments, " low palmitic acid content " by the feature palmitic acid content with wild-type parent kind into Row is relatively determined.Therefore, the first sunflower comprising low palmitic acid content phenotype is compared with Wild-type sunflower, Huo Zheyu The parent's Sunflower Varieties for generating first sunflower are compared, and can have the palmitinic acid of " reduction " or " reduction " level.It reduces It is opposite term with reducing, shows that plant generates or containing palmitinic acid more less than similar wild-type plant.

Sunflower plants palmitic acid content changes greatly, and the feature palmitic acid content performance measured in certain species For a series of high and low palmitic acid content phenotypes.However, can determine different plants, plant lines or plant by simply observing The opposite palmitic acid content of object family.Moreover, Sunflower Varieties show the genetically confirmable of " palmitic acid content " Phenotype grade.Those skilled in the art is familiar with for quantitative and the sunflower plants palmitic acid content that scores measuring method.Plant Palmitic acid content the various conventional analytical techniques of this field can be used quantified, such as but be not limited only to, NMR；NIR； And soxhlet's extraction.

The verification of low palmitic acid content can by using or be applicable in existing palmitic acid content method and realize.For example, NMR, NIR, and/or soxhlet's extraction can be used for verifying low palmitic acid content character in any specified plant or group still with spy Calibration note separates together.In some embodiments, these and other methods can be used also to measure by will be with low palm The degree that the palmitic acid content that the chain label of acid content penetrates into or recombination is introduced into desired genetic background and realizes reduces.

IV. the low palmitic acid content label in sunflower

Embodiment of the present invention includes the label chain with palmitic acid content low in sunflower.These labels can be used In, such as but be not limited only to, identification have it is higher may include low palmitinic acid phenotype sunflower plants or germplasm；It selects this kind of Sunflower plants and germplasm (for example, in marker assisted selection program)；Not having more high likelihood with identification and selection includes The sunflower plants and germplasm of low palmitinic acid phenotype.Compared with this field currently existing composition and method, this paper institute is used The one or more labels stated can provide in terms of time, cost and the labour in sunflower breeding for plant breeder excellent Gesture.

Disclosed herein is the regions low palmitic acid content QTL for being accredited as being located in sunflower genome linkage group 5 (LG5) Interior or neighbouring specific markers, they are polymorphism in parent genotype.In these QTL label, there is specific label Allele palmitic acid content phenotype low with sunflower is chain.In some embodiments, palmitic acid content table low with sunflower The chain QTL of type is marked selected from the label subgroup provided from Fig. 1.Such as it but is not limited only to, palmitic acid content low with sunflower The chain QTL label of phenotype is selected from HA0031B；HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870； HA0907；And HA0612A.

Using target group's determination and the chain label of low palmitic acid content.In some embodiments, such fixed Position group can come from hybridization H757B/H280R, although other groups can also alternatively use.Many appropriate software platforms Any can be used for determining chain marked locus.Such as but be not limited only to,；；WithIt can be used in specific example.In some embodiments, such as when in linkage analysis When middle use software, reflect that the data of allelic information detected can passed during use or using preceding by electronics Defeated or Electronic saving, such as store in computer-readable medium.

In some embodiments, may comprising low palmitic acid content phenotype the first sunflower plants or germplasm be to pass through Detect multiple marker alleles identification in first sunflower plants or germplasm.Such as but be not limited only to, it is specific real The scheme of applying include for identify may comprising low palmitic acid content phenotype plant or germplasm method, wherein from molecular labeling HA0031B；HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870；HA0907；With in HA0612A detection with it is low The chain marker allele of palmitinic acid.It, may be comprising low palmitic acid content phenotype for identifying according to some embodiments The method of plant or germplasm includes from molecular labeling HA0031B；HA0908；HA1665；HA0304A；HA0850；HA0743； HA0870；HA0907；With more than one marker allele chain with low palmitinic acid of detection in HA0612A.It is specific to implement Scheme include for detect may comprising low palmitic acid content phenotype plant or germplasm method, wherein from be selected from HA0031B；HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870；HA0907；With HA0612A at least one Marker allele is detected in the chain molecular labeling of low palmitinic acid linked marker.

In some embodiments, detected allele is and the positively related allele shape of low palmitic acid content Formula.Alternatively, detected allele can be the allelic form with low palmitic acid content negative correlation, in such case Under, allele can be by counter-selection.Select more than a marker allele for detect in the case where, for each label Select allele；Therefore, two or more allele are detected.In some instances, label may include more than one having (such as positively related) allelic form of benefit；In such example, these advantageous allelic forms be can detecte Any one.

Therefore, multiple marker alleles can be detected simultaneously in single plant, germplasm or plant population.In this side In the example of method, such plant or germplasm can choose, contain from the more than one and chain mark of low palmitic acid content The positive correlation allele of note.It, can will be from more than one with the chain label of low palmitic acid content in specific example Positive correlation allele penetrate into target (such as receptor) sunflower germplasm.Those skilled in the art can be appreciated that, Simultaneous selection (and/or gene transgression) is positively related etc. from more than one low palmitic acid content label in identical plant or germplasm Position gene may generate additivity (such as collaboration) phenotype in plant or germplasm.

Although specific marker allele may be isolated with low palmitic acid content phenotype, these marked locus It is not necessarily a part for contributing to the QTL locus of (such as causing) low palmitic acid content.For example, not requiring to isolate label In the gene for contributing to or assigning low palmitic acid content (such as a part as the gene open reading frame).It is specific Being associated between marker allele and low palmitic acid content phenotype may be by the low palmitic acid content allele institute It is original between the marker allele isolated and the low palmitic acid content allele of QTL in ancestors' Sunflower Lines of origin Caused by " coupling " (coupling) chain phase.Finally, by recombinating repeatedly, the exchange between label and QTL locus is isolated Event may change this orientation.Positively related marker allele may change in the successive generation as a result, this takes Certainly in for creating chain phase present in the low palmitic acid content parent of segregating population.The fact that will not reduce the label Monitor the use of phenotype separation；It is positively related which marker allele form it, which can only change in given segregating population, (relatively, negatively correlated).

When two genetic elements of instruction (such as the genetic elements for contributing to low palmitic acid content and immediate mark Note) between relationship when, " coupling " chain phase refer to positives correlation allele low palmitic acid content QTL at it is respective chain Situation of the positive correlation allele physical interconnection of marked locus on identical chromosome chain.In coupling phase " in, two equipotentials The offspring that gene is inherited the chromosome chain together inherits.In " repel each other (repulsion) " is mutually chain, gene of interest seat The equipotential of the usual negative correlation being positively correlated at allele and neighbouring marked locus at (such as QTL of low palmitic acid content) Gene physical linkage, therefore two usual positively related allele will not be inherited together (that is, the two locus are each other " different phases (out of phase) ").

As it is used herein, " positive correlation " allele of label is the allele of the label in positioning described herein In group with desired phenotype (for example, the low palmitic acid content) person of isolating.However, it is contemplated that above it should be appreciated that due to repelling phase A possibility that chain, can may equivalently use other equipotential bases of label in the embodiment that other are related to different groups Because of form.

Similarly, the linked marker allelic form not isolated with low palmitic acid content can also be alternatively some It is used in embodiment, because this allelic form can be used for identifying the plant that can not have low palmitinic acid phenotype.Example Such as, this allele can be used for the purpose (such as counter-selection) excluded in breeding, negative with low palmitic acid content with identification Relevant allele, and/or high palmitic acid content plant or germplasm are eliminated subsequent breeding round.

One QTL label at least has a positively related allele, although in some instances can be in group It was found that QTL, which is marked with two or more, is positively correlated allele.Any positive correlation allele of this label can be used for, example Such as, identify and construct low (such as reduction) palmitic acid content Sunflower Lines.In some instances, identified in plant (or Penetrated into plant) 1,2,3 or more positively related allele of the not isolabeling chain with low palmitic acid content, and And positive selection or counter-selection can be carried out to all these positive correlations label or its subset during MAS.In some embodiment party In case, at least one plant or germplasm are identified, has at least one such positively related with low palmitic acid content phenotype Allele.

Marked locus itself is exactly character, therefore can be analyzed it according to conventional linkage analysis, such as pass through Marked locus is tracked during the separation.Therefore, in some embodiments, chain between measurement label, for example, 1cM is equivalent to the first marked locus, and by exchanging, (it can be any other character with the second locus in an independent generation (for example, second marked locus) or another include or include the trait locuses in QTL) separated chance is 1%.

When the genetic marker that marks (QTL that example provides as shown in figure 1 label and its equivalent) chain with QTL and given QTL label is substantial access to (that is, abundant close linkage), when so that the genetic marker and QTL label showing low recombination frequency, these Genetic marker is particularly useful.In some embodiments, linked marker and QTL label display about 10% or lower are heavy Group frequency (that is, given label is within about 10cM of QTL).According to definition, these feelings of linked gene seat at least 90% It can be isolated under shape.Really, label marks closer with QTL, marks the indicant as anticipant character more effective and advantageous.So And at a distance from QTL be more than such as 10cM label be also likely to be it is useful, especially when being combined with other linked markers.

Therefore, in some embodiments, chain locus, such as QTL marked locus and the second marked locus Recombination frequency between display about 10% or lower locus；Such as it but is not limited only to, about 9% or lower, about 8% or more It is low, about 7% or lower, about 6% or lower, about 5% or lower, about 4% or lower, about 3% or lower, and it is big About 2% or lower.In some instances, related gene seat (such as marked locus and target gene seat, such as QTL) display is about 1% or lower recombination frequency；Such as but be not limited only to, about 0.75% or lower, about 0.5% or lower, and about 0.25% or lower.Therefore, in specific embodiments, each locus can be separated by about 10cM；About 9cM；About 8cM； About 7cM；About 6cM；About 5cM；About 4cM；About 3cM；About 2cM；About 1cM；About 0.75cM；About 0.5cM； About 0.25cM；Or it is less.In some instances, specific linked marker can be by checking the genetic map of sunflower genome (such as including LG5 person) is determined.

In certain aspects, chain to be expressed as the recombination frequency limit, or it is expressed as heredity or physical distance range.Example Such as, in some embodiments, two chain locus are two and are separated by the locus less than 50cM map unit.Some In example, chain locus is two locus being separated by less than 40cM.In some instances, two linked gene seats are two A locus being separated by less than 30cM.In some instances, two linked gene seats are two locus being separated by less than 25cM. In some instances, two linked gene seats are two locus being separated by less than 20cM.In some instances, two chain bases Because seat is two locus being separated by less than 15cM.In some instances, the chain range that can be expressed as upper and lower bound； Such as it but is not limited only to, about 10-20cM；About 10-30cM；About 10-40cM；About 0.5- about 10cM；About 0.1- About 9cM；About 0.1- about 8cM；About 0.1- about 7cM；About 0.1- about 6cM；About 0.1- about 5cM；About 0.1- about 4cM；About 0.1- about 3cM；About 0.1- about 2cM；About 0.1- about 1cM；About 0.1- is about 0.5cM。

In some embodiments, label as described herein (for example, label those of is shown in FIG. 1, HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and with it is above-mentioned at least one Chain label) it is positively correlated with low sunflower palmitic acid content.Therefore, these label may with low palmitic acid content QTL and/or Character is substantial access to, so that one or more of these labels can be used as prediction of low palmitic acid content character.It is this pre- Survey ability is exceedingly useful in MAS environment, as will be discussed in greater detail herein.

It is as described herein to mark the use of chain label to be not restricted to appoint with low palmitic acid content phenotype and/or QTL What specific sunflower genetic map or methodology.It should be noted that the list of linked marker is in figure and figure and side due to various factors It can be different between the science of law and methodology.For example, the label being located on any two figure may not be identical, and a figure may There is bigger mark density than another figure.It equally, can not for constructing target group, methodology and the algorithm of genetic map Together.It will be understood by those skilled in the art that a genetic map is not necessarily more accurate or more inaccurate than another, and technology Personnel will also be recognized that any sunflower genetic map is used equally for the determining and chain label of specific markers.For example, specific chain Label can be determined from any genetic map (such as lab diagram or integration map) known in the art, and can be from any new Location data collection is determined.

Embodiment of the present invention is not limited only to any specific Helianthus annuus population or the (example using any particular methodology Such as, any specific software or any specific software parameter group) identify or determine specific markers and low palmitic acid content table Type it is chain.According to the disclosure, the feature of label as described herein will be extrapolated to any sense by those skilled in the art The sunflower gene pool of interest or group, and any specific software and software parameter can be used in doing so.

V. the detection of the low palmitic acid content label in sunflower

For detect (identification) carry low palmitic acid content marked locus specific allele sunflower plants or The method of germplasm is the feature of some embodiments.In some embodiments, available a variety of label detection sides in this field Any one of method may be used to detect marker allele, this depends on the type for being detected label.In instances, it is used for The appropriate method of label detection may include amplification and identify that resulting amplification marks, by, for example, but be not limited only to, PCR； LCR；With the amplification method (such as ASH, SSR detection, rflp analysis and many other methods) based on transcription.

Generally, the detection of genetic marker relies on one or more properties of nucleic acid.For example, some for detecting hereditary mark The technology of note (such as uses sunflower genomic DNA molecule as template using the corresponding nucleic of probe nucleic acid and genetic marker The amplification of nucleic acid of generation) hybridization.In certain embodiments, can be used including, such as but be not limited only to, solution phase, Solid phase, the hybridization format of mixed phase and in situ hybridization measurement carry out allele detection.Detailed guidance about nucleic acid hybridization can To be found in such as following documents: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Elsevier,NY。

Label corresponding to the genetic polymorphism between group member can be detected with any one of a variety of methods, these Method for example but is not limited only to, the method based on nucleic acid amplification；With the nucleotide sequencing in polymorphism mark area.Many detection methods (method including the sum based on amplification based on sequencing) can easily be adapted to high throughput analysis in some instances, Such as by using available high-flux sequence method, such as sequencing by hybridization.

Amplimer for expanding SSR- phenotypic marker locus is included in the particular instance of some embodiments.Table 6 Provide the specific primer for expanding specific markers as described herein.However, technical staff can be immediately appreciate that, give The other sequences of primer either side can replace given primer, as long as it includes the allele to be detected that the primer, which can expand, Nucleotide sequence.Moreover, the specific probe for allele detection can change.For example, any can identify is wanted The probe in the region of the label amplicon of detection can replace the exemplary probe listed herein.Moreover, amplimer and detection The configuration of probe also can change.Therefore, embodiment is not limited in the primer and probe specifically listed herein.Although herein Many specific primer examples (being shown in Table 6) are provided, but any suitable method can be used designs the present invention and can make Proper probes.For example, any suitable software program can be used, such as but it is not limited only to, design Primer of equal value.

Molecular labeling can be detected with the available establishment method in this field, and such method includes for example but not only limiting In: ASH or other methods for being used to detect SNP；AFLP detection；The variable sequence of amplification detects；RAPD detection；RFLP detection； From maintenance sequence replicating detection；SSR detection；SSCP detection；It is detected with isoenzyme mark.Although the example provided in Fig. 1 and table 6 Label is SSR marker, but any in foregoing tags type can be with identifying comprising tribute in certain embodiments It offers in the chromosome segment of the genetic elements of the low palmitic acid content phenotype of sunflower.

For example, the label comprising RFLP can (it be usually subfragrnent by, for example, the probe for making the nucleic acid to be detected Or synthetic oligonucleotide corresponding to subfragrnent) detected with the genomic DNA hybridization of limitation enzymic digestion to be detected.Choosing Restriction enzyme is selected to be provided at least two in Different Individual or group and the restriction fragment of (or polymorphism) length may be selected.Really Determine one or more to be a simple process for the restriction enzyme for the segment that each hybridization generation provides information, provide After target DNA sequence, it can easily be realized by those skilled in the art.In suitable matrix (such as agarose or polypropylene Amide) in separated and be transferred on film (such as nitrocellulose or nylon) based on length after, probe can led to Hybridize labeled probe under conditions of being combined with target balance, extra probe is then removed by cleaning, and detect and marked The probe of note.

In some embodiments, use amplification step as detection marked locus/to marked locus carry out gene A part of the method for parting.However, amplification step is not all to be in all cases necessary to label detection.For example, not The genomic DNA of amplification can be simply detected and carrying out Southern trace to genome DNA sample.Amplification/ Also can be omitted individual detection probe in detection method, this can for example, by but be not limited only to following manner and realize: it is real Real-time amplification reaction is applied, real-time amplification reaction detects the formation of product by the modification of amplimer when incorporation product；It will be by The nucleotide of label is incorporated into amplicon；With the change by monitoring amplicon molecular rotation property compared with the precursor not expanded Change (such as by fluorescence polarization).

PCR, RT-PCR, real-time PCR and LCR by particularly widely be used as amplification and amplification detection method, for expand and It detects nucleic acid (such as nucleic acid comprising marked locus).About using the details of these and other amplification method can be a large amount of It is found in any in standard textbook, including for example: Sambrook etc., Molecular Cloning:A Laboratory Manual (2000) third edition, 1-3 chapter, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY；Current Protocols in Molecular Biology (augments) F.M.Ausubel etc. from version in 2002 and compiles, Current Protocols, Greene Publishing Associates company and John Wiley&Sons company are joint； It is compiled with PCR Protocols A Guide to Methods and Applications (1990) Innis etc., Academic Press Inc.,San Diego,CA.Additional detail about the detection of plant amplifying nucleic acid is also shown such as Plant Molecular Biology (1993) Croy (eds.) BIOS Scientific Publishers, Inc.

It can also be found in such as following documents and execute amplification in vitro and detection method about being enough guidance technology personnel, It is mediated including polymerase chain reaction (PCR), ligase chain reaction (LCR), Q β-duplication enzymatic amplification and other RNA polymerases Technology (such as NASBA) and the example technology additional detail: United States Patent (USP) 4,683,202；Arnheim and Levinson(1991)J.NIH Res.3:81-94；Kwoh et al.(1989)Proc.Natl.Acad.Sci.USA 86: 1173；Guatelli et al.(1990),supra；Lomell et al.(1989)J.Clin.Chem.35:1826； Landegren et al.(1988)Science 241:1077-80；Van Brunt(1990)Biotechnology 8:291- 4；Wu and Wallace(1989)Gene 4:560；Barringer et al.(1990)Gene 89:117；With Sooknanan and Malek(1995)Biotechnology 13:563-4.By the modification method of PCR amplification large nucleic acids, It can be used in some applications of positional cloning, in Cheng et al. (1994) Nature 369:684 and its reference There is further description in bibliography, wherein can produce PCR amplification up to 40kb.

Many available biological textbooks also further discussing about PCR and related amplification method.Technical staff's meeting , it is realized that substantially any RNA, which can be transformed into, is suitable for restrictive digestion, PCR amplification and with reverse transcriptase and polymerase The double-stranded DNA of (such as passing through RT-PCR) is sequenced.

In some embodiments, nucleic acid probe can be used for detecting the nucleic acid comprising marker allele nucleotide sequence. These probes can be for example, for separating and the chain nucleotide sequence of marker allele sequence in positional cloning.In spy Determine nucleic acid probe useful in embodiment not limited by the constraint of any specific dimensions.In some embodiments, nucleic acid is visited The length of needle can be, such as but be not limited only to, at least 20 nucleotide；At least 50 nucleotide；At least 100 nucleotide； At least 200 nucleotide.It can be clone's and/or synthesis for the nucleic acid probe of marked locus.

Any suitable marker can be used together in specific example with probe.It is suitable for making together with nucleic acid probe Detectable marker include it is any can by light splitting, radioactive isotope, photochemistry, biochemistry, immunochemistry, The component of electricity, light or chemical means detection.Therefore, hybridization probe can be used, for example, autoradiograph, fluorography or other Similar detection technique detection, this depends on particular marker detected.Useful marker includes biotin (for using band The Streptavidin conjugate of marker dyes), magnetic bead, fluorescent dye, radioactively labelled substance, enzyme and colorimetric marker.Other Marker includes the ligand in conjunction with the antibody or specific binding target that are marked with fluorogen, chemiluminescent agent and enzyme.Probe is also It may include being used to generate with radiolabeled amplicon with radiolabeled PCR primer.About for labeling nucleic acid The additional information of labelling strategies and corresponding inspection policies can be found in such as following documents: Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals,Sixth Edition,Molecular Probes,Inc.,Eugene OR；With Haugland (2001) Handbook of Fluorescent Probes and Research Chemicals, Eighth Edition, Molecular Probes, Inc., Eugene, OR (have CD ROM can With).In particular instances, PCR is detected and is quantitatively used the fluorogenic oligonucleotide probe with double labelling object, such as Probe (Applied Biosystems) is implemented.

In some embodiments, primer is without marker, and marks PCR amplification can be for example, size parses (size resolution) is visualized afterwards (such as after agarose gel electrophoresis).In particular instances, it is parsed in size Afterwards to the ethidium bromide staining of PCR amplification allow will correspond to different marker alleles various sizes of amplicon it is visual Change.

Primer used in embodiment is not limited only to that the primer of the amplicon of specific dimensions can be generated.For example, being used for The primer of amplification expression of specific marker genes seat and allele is not limited only to the primer of amplification related gene seat whole region.Primer can To generate the amplicon of any appropriate length, length than allele define in provide it is longer or shorter.In instances, it marks The length for the amplicon that note amplification can produce is, such as but is not limited only to, at least 20 nucleotide；At least 50 nucleotide； At least 100 nucleotide；At least 200 nucleotide.

For make oligonucleotides and comprising oligonucleotides useful composition (for example, probe, primer, molecular beacon, PNA and LNA) synthetic method be well known to the skilled person.For example, oligonucleotides can be according to for example Beaucage and Caruthers (1981) Tetrahedron Letts.22 (20): solid phase phosphorous described in 1859-62 Amide triester method chemically synthesizes.These methods can be using the synthesizer of automation, such as but is not limited only to, Needham- Described in VanDevanter et al. (1984) Nucleic Acids Res.12:6159-68.Oligonucleotides (including By the oligonucleotides of modification) it can also be ordered from many commercial sources, including for example but be not limited only to, The Midland Certified Reagent Company；The Great American Gene Company；ExpressGen Inc.；With Operon Technologies Inc.Similarly, PNA can be customized from any source in many sources, including for example but not It is only limitted to, PeptidoGenic；HTI Bio-Products,Inc.；BMA Biomedicals Ltd(U.K.)；With Bio.Synthesis,Inc。

In some embodiments, method (in silico method) the detection label etc. simulated on calculating can be used Position gene.For example, the nucleic acid sequence comprising flag sequence can store in a computer.Desired marked locus sequence (or Its homologue) it can be identified with suitable nucleotide searches algorithm, for example, by for example but being not limited only to,It mentions The algorithm of confession or simpler word processor.

In some embodiments, marker allele is detected using the detection method of based on PCR, wherein including mark The size or sequence of PCR amplification of note can indicate the existence or non-existence of specific markers allele.In some instances, PCR primer hybridizes with the conserved region of polymorphism mark area two sides.PCR primer for such amplifier molecule label is in the art Sometimes referred to as " PCR label " (PCR markers) or referred to as " mark ".

The mainspring that molecular labeling is developed in crop is to improve plant breeding by marker assisted selection (MAS) The potentiality of efficiency.It can be used for identifying with the genetic marker of character interested or gene linkage and contain at one or more locus It is expected that the plant of marker allele, it is therefore contemplated that these plants can be by the expectation marker allele and the interested property Shape or gene are transferred to their offspring together.Genetic marker can be used for identifying in a locus or several non-chain or Contain the plant of specific genotype at chain locus (for example, haplotype).Similarly, as being intended to improve sunflower yield The whole MAS procedure of breeding a part, marker allele as described herein can be penetrated into any desired sunflower In genetic background, germplasm, plant, strain, kind etc..

According to some embodiments, it is as described herein label provide identification comprising low or reduction palmitic acid content (or Palmitic acid content that is high or increasing) sunflower plants and germplasm means, it is to include example at some locus by identification Such as HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, the specific equipotential of HA0907, HA0612A Gene, and realize with the plant of at least one chain label above-mentioned and germplasm.Lacked and low palmitinic acid by identification The plant for the marker allele that content isolates can identify that (or palmitic acid content reduces for high palmitinic acid plant and germplasm Obtain less plant), such as to eliminate it from subsequent hybridization and breeding.

According to above, embodiment of the present invention includes having and contributing to or assign low (such as reduction) palmitinic acid and contain The molecular labeling for the significant probability that the QTL of scale type is isolated.These QTL label can be in anticipant character (reduced palm Acid content) marker assisted selection in be applied, and there are other purposes.Embodiment of the present invention is not limited in appointing What is for being checked or analyzed the ad hoc approach of these labels.

VI. low palmitic acid content marker gene is penetrated into Sunflower Varieties

As previously mentioned, identification includes and low (such as reduction) palmitic acid content phenotype chain marker allele Sunflower plants or germplasm, for implement sunflower marker assisted selection provide the foundation.In some embodiments, it selects At least one contains the sunflower plants of at least one and the positively related marker allele of low palmitinic acid, while can be with counter-selection Sunflower plants comprising the marker allele with low palmitic acid content negative correlation.

It can be specific (such as excellent or outer to having with gene transgression with the positively related expectation marker allele of low palmitinic acid Come) in the sunflower of genetic background, to generate the low palmitic acid content sunflower plants or germplasm of gene transgression.In some realities It applies in scheme, multiple low palmitic acid content labels can established sequentially or simultaneously be selected and/or gene transgression is into sunflower.It can be with There is no limit for the specific combination of the low palmitic acid content label selected in single plant or germplasm, and can include label, Example those of proposition as shown in figure 1, any label chain with label listed in Fig. 1 or any positioned at the area QTL as defined herein The combination of interior label.

In embodiments, it identifies and the positively related QTL marker allele of low palmitic acid content of sunflower plants Ability also for selection there is the plant of advantageous marked locus to provide method.It any is accredited as wrapping for example, can choose Plant containing desired marked locus (such as with the positively related marker allele of low palmitic acid content), and lack should for counter-selection The plant (or plant comprising the allele with low palmitic acid content negative correlation) of allele.Therefore, in particular implementation In scheme, in the first plant or germplasm after identification marking allele, gene transgression method includes the first sunflower of selection Plant or germplasm, or select the offspring of the first plant or germplasm.In some instances, can by it is resulting be selected to day Certain herbaceous plants with big flowers plant or germplasm and the second sunflower plants or germplasm (such as breeding sunflower or external sunflower) hybridization, to generate packet Offspring containing the marker allele and desired characteristic and/or the second plant or germplasm allele.

In some embodiments, the method for the low palmitinic acid QTL of gene transgression may include, for example, provide at least one with The chain label of low palmitinic acid (for example, the label isolated with low palmitinic acid)；In the first plant comprising low palmitinic acid QTL or Marker allele is determined in germplasm；With by marker allele gene transgression to the second sunflower plants or germplasm, thus Generate the sunflower plants or germplasm of gene transgression.In certain embodiments, the second sunflower plants or germplasm can wrap Containing palmitic acid content increased compared with the first sunflower plants or seed, and the sunflower plants of gene transgression or germplasm will wrap Containing the palmitic acid content reduced compared with the second plant or germplasm.As hereinafter will be discussed in greater detail, implemented by these The sunflower plants or germplasm for the gene transgression that scheme and other embodiments generate are also contained in embodiment of the present invention.

In some embodiments, wherein the sunflower plants of gene transgression or germplasm pass through any side provided in this article Method generates, and the sunflower plants or germplasm of gene transgression can be formed with the fatty acid of the oil in the seed of the plant come table Sign.The plant of gene transgression or germplasm may include in the seed oil from the plant, such as but be not limited only to, about 3% Or less palmitinic acid.In some embodiments, the sunflower plants of this gene transgression or germplasm are in the seed from plant Include about 2.5% or less palmitinic acid in oil, such as but is not limited only to, 2.6%；2.5%；2.4%；2.3%；2.2%； 2.1%；2.0%；1.9%；1.8%；About 1.7%；It is lower.

In addition to the marker allele selected is penetrated into (such as passing through conventional breeding methods) into desired genetic background To by low palmitinic acid QTL gene transgression into the background except, transgenic method can also be used in some embodiments Generate the sunflower plants and/or germplasm of low palmitic acid content.It in some embodiments, can will be in sunflower at least one The chain exogenous nucleic acid (such as gene or open reading frame) of a label as described herein is introduced into target plant or germplasm.Example Such as, can be cloned from sunflower genomic DNA with it is as described herein at least one mark chain nucleic acid coding sequence (such as Pass through positional cloning), and be introduced into target plant or germplasm.

Therefore, specific embodiment includes generating the side of the sunflower plants comprising low palmitic acid content phenotype or germplasm Method, wherein this method includes introducing exogenous nucleic acid in target sunflower plants or its offspring, wherein the exogenous nucleic acid and as follows Nucleotide sequence it is substantially the same, the nucleotide sequence and one or more with the chain marker gene of low palmitic acid content At least one positively related marker allele at seat is chain.In some instances, marked locus can be selected from: HA0031B； HA0908；HA1665；HA0304A；HA0850；HA0743；HA0870；HA0907；HA0612A；With with it is above-mentioned at least one The label of chain (for example, showing no more than 10% recombination frequency).In some embodiments, multiple linked markers can be used Construct genetically modified plants.In this multiple label using label described herein which by practitioner's discretion.

Any of a variety of methods is used equally for providing exogenous nucleic acid for sunflower plants or germplasm.In some embodiments In, by positional cloning isolated nucleic acid sequence, and by with the company with the positively related marker allele of low palmitic acid content Lock is identified.For example, the nucleotide sequence can correspond to encode the open reading frame (ORF) of following polypeptide, the polypeptide It will lead to when being expressed in sunflower plants or facilitate sunflower plants that there is low palmitic acid content.The nucleotide sequence can be with Group enters into exogenous nucleic acid molecule afterwards.The exact composition of exogenous nucleic acid can change.For example, exogenous nucleic acid may include that expression carries Body, so as to nucleotide sequence described in the plant interior expression that is imported in the exogenous nucleic acid.

Chain label can use the method comprising marker assisted selection and penetrate into (for example, whereby with low palmitic acid content Penetrate into low palmitic acid content phenotype) into sunflower plants or germplasm.In embodiments, using be accredited as have with it is low The polymorphism mark of the significant probability that palmitic acid content character isolates implements MAS.These labels in Fig. 1 (for example, list Those of) it is estimated to be the gene for being located in and contributing to the low palmitic acid content of plant (compared with the plant comprising wild type gene) Close to or within.These labels can be counted as instruction of character, and can be referred to as QTL label.In embodiments, it plants Object or germplasm are tested the presence that allele is positively correlated at least one QTL label.

In embodiments, it is statistically significant to determine which polymorphism mark allele shows using linkage analysis The likelihood isolated with low palmitic acid content phenotype.Identifying such and low positively related label of palmitic acid content phenotype After allele, the label can be used and quickly and accurately screen low palmitic acid content allele in plant lines, By its life cycle and phenotypic assessment is waited without planting plants.Moreover, the identification of label allows to specific low palm Acid content allele carries out hereditary selection, even the situation unknown in the molecular identity of the practical low palmitic acid content QTL Under.A small tissue samples can be obtained from the offspring's sunflower plants generated by hybridization (for example, from first of plant Leaf) and screened with suitable molecular labeling.Whereby, it can quickly determine whether offspring should continue further to educate Kind.Linked marker also eliminated the influence that may influence the environmental factor of phenotypic expression, to allow in such a way that environment is neutral Select the sunflower of low palmitic acid content.Therefore, although various environmental factors may seem surface to the contribution of vegetable oil character On can perplex the use of label described herein, but peculiar advantages for actually these labels are exactly that their availability is disobeyed Rely in environment.

In some embodiments comprising MAS, polymorphism QTL marked locus can be used select containing with low palm The plant of the positively related marker allele of acid content phenotype.For example, can be in the biological sample from the plant to be selected Detect the corresponding nucleic of labeling nucleic acid allele.The detection can be using probe nucleic acid and marker allele or its amplicon Hybridization form (for example, using allele specific hybridization, Southern analysis, Northern analysis, in situ hybridization, and Primer hybridization then carries out PCR amplification to the region of label).There are (or being not present) specific markers etc. in verifying biological sample After the gene of position, plant is selected, and can be used for manufacturing progeny plants by selection and use in some instances.

Sunflower plants breeder it is expected low palmitic acid content marker gene seat and other anticipant characters (such as high yield Rate) the combination of label/gene, with the Sunflower Varieties of development and improvement.Pass through non-molecular method (such as the property of sunflower plants Shape assessment) a large amount of samples of screening are usually costly, time-consuming and insecure.Use as described herein and low palmitic acid content QTL Chain polymorphism mark provides a kind of effective method to select desired kind in the procedure of breeding.Low palmitinic acid contains Advantage of the marker assisted selection of amount compared with field evaluations includes, for example, MAS can be carried out in 1 year any time, and It need not consider the season of growth.Moreover, as previously proposed, environment influences substantially unrelated with marker assisted selection.

When a group (such as contains relative to multiple marked locus with one or more linkages of characters with low palmitinic acid Measure chain multiple labels) separation when, the efficiency of MAS with phenotypic screen compared with understand it is bigger because all marked locus can To be evaluated together from single DNA sample in the lab.In specific embodiments of the present invention, can from single sample or HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743 are concurrently or sequentially measured from multiple parallel samples, HA0870, HA0907 and HA0612A label, and at least one aforementioned chain label.

Another purposes of MAS in plant breeding, which is to aid in, recycles circulation parent genotype by back cross breeding.It carries out The purpose of backcrossing is usually by one or a few label or QTL locus from donor parents (such as include desired low palm fibre The parent of palmitic acid acid content marked locus) penetrate into other from recurrent parent (for example, other high yield Sunflower Lines) In desired genetic background.The wheel number of the backcrossing carried out is more, hereditary tribute of the recurrent parent to resulting gene transgression kind It offers bigger.In some instances, the backcrossing of many wheels can be can be carried out, this is because for example, low palmitic acid content plant may be by In other reasons (such as low-yield, low fertility etc.) be undesirable.Relatively, the strain generated in Intensive breeding program may With excellent yield, fertility etc., and only at a desired aspect, for example, on palmitic acid content it is defective.It is coming from The specific markers in donor source (it may be the excellent genetic background that may also be not intended to serve as the excellent variety of circulation strain) In label auxiliary backcrossing, practitioner can select donor label in backcross progeny, then utilize and return repeatedly with circulation strain It hands over to rebuild the genome of circulation strain as much as possible.

According to aforementioned, label as described herein and method can use to instruct people to be had and superior agronomy It can (for example, low palmitic acid content, and to any other available label about yield, disease resistance etc.) relevant dyeing The marker assisted selection or breeding of the Sunflower Varieties of the expectation complement (group) of body segment allelic form.It can will be any The marker allele is introduced into Sunflower Lines by gene transgression (such as passing through traditional breeding method and/or conversion), To generate the sunflower plants with superior agronomy performance.In some embodiments, if the nucleic acid from plant is to the phase The genetic marker allele of prestige be it is positive, then plant can with self-pollination to create the pure lines with phase homogenic type, or Person can by they with comprising same tag allele or it is other expectation label and/or feature plant hybridization it is sexual to create The hybrid generation of hybridization.

Many times, by method of the invention be applied at least one relationship sunflower plants, such as from theme to Ancestors' strain or lines progeny in day certain herbaceous plants with big flowers plant pedigree, so as to track the something lost of desired low palmitic acid content allele Convey feelings condition.The algebra that each sunflower plants for the method for the present invention are separated by is generally 1-20 generation, usual 1-5 generation, typically It was separated by for 1,2 or 3 generation, very often, the direct filial generation of sunflower plants or parental generation are used for this method (that is, every 1 generation).

Genetic diversity is important in breeding project.In the limited situation of diversity, when all advantageous equipotentials When gene is fixed in excellent group, the genetic gain obtained in the procedure of breeding will be eventually reached platform.Therefore, plant educates One purpose of kind is diversity to be introduced in excellent pond without losing the genetic gain having been achieved with, and use least possibility Investment.Which genome area MAS provides and which favorable allels from original ancestry have been subjected to selection and with The conservative instruction of time passage, to help to introduce from Exotic Germplasm Resources (parent with excellent genes pond without relationship) advantageous It makes a variation to find the effort for the favorable allels being not currently present in excellent genes pond.Therefore, in some embodiments In, the MAS as described herein marked in the hybridization that can be used for being related to (excellent x is external) Sunflower Lines, by separation offspring MAS is carried out, to maintain the low palmitic acid content marker allele of main yield allele and this paper.

Can be used in some embodiments molecular marker gene seat and allele as described herein (such as HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and with it is above-mentioned At least one chain label), as previously mentioned, may then pass through well known method will to identify low palmitic acid content QTL QTL clone.These low palmitic acid contents clone can genetic linkage first by it with label as described herein be subject to Identification.For example, " gene location clone " is contained to define containing low palmitinic acid using the close physical proximity that low palmitic acid content marks Measure the separation chromosome segment of QTL gene.Isolated chromosome segment can be generated by well-known method, such as but not It is only limitted to, with one or more limitation enzymic digestion chromosomal DNAs, with PCR amplification chromosomal region and any suitable alternative expansion Increase reaction.Then the load for being suitable for replicating and/or express institute's Insert Fragment will can be connected to by the segment for digesting or expanding In body.The label adjacent to ORF relevant with phenotypic character can be with DNA clone (for example, gram from genome dna library It is grand) specific hybrid, thus identify the clone for having the ORF (or segment of ORF) thereon.If label and low palmitic acid content Farther out, then the segment containing the ORF can be identified the distance of QTL gene by multi-turns screen to clone and separation, this A little clones form one section of continuous DNA sequence dna altogether.This process is commonly referred to as " chromosome walking ", and it can be used In generation " contig (contig) " or " contig map ".

It can find in the following documents and be enough the method that guidance technology personnel separate clone relevant to linked marker: example Such as, the (eds.) such as Sambrook Molecular Cloning:A Laboratory Manual, the second edition, the 1-3 volumes, Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,1989；It is compiled with Ausubel etc., Current Protocols in Molecular Biology, the 2nd chapter, Greene Publishing and Wiley- Interscience,NY,1995。

VII. the plant comprising low palmitic acid content label

Some embodiments include the method for making sunflower plants, and further comprise these sunflower plants from Body.In certain embodiments, this method may include making the with the chain mark of low palmitinic acid as described herein One parent's sunflower plants hybridize with the second sunflower plants, wherein the first sunflower plants include at least one and low palmitinic acid Positively related marker allele；With female sunflower plants are cultivated under plant growing condition, to generate sunflower plants Offspring.Can be to these sunflower plants offspring analysis and the chain marker allele of low palmitic acid content, and optional select a time The offspring of prestige.These progeny plants or its seed can be used for various uses, include but are not limited to, can with commercial distribution they It is produced for sunflower；For food；Processing obtains desired sunflower product (for example, sunflower oil)；And/or in subsequent rounds It is further used in secondary breeding.Sunflower plants according to some embodiments include at least one containing label described herein The progeny plants of a allelic form, so that further offspring be enable to inherit the marker allele.

It includes low palmitic acid content (for example, reduced palmitic acid content) to day that some embodiments, which include for generating, The method of certain herbaceous plants with big flowers plant.In certain embodiments, these methods may include by conventional plant breeding or by by external source DNA (such as transgenosis), which is introduced into sunflower cultivar or plant, generates this plant.

Therefore, some embodiments include the host cell with the nuclear transformation corresponding to low palmitic acid content QTL and life Object, wherein QTL is using at least one Marker Identification chain with low palmitic acid content as described herein.In some examples In, these nucleic acid may include chromosome interval (such as the gene for the expression product that coding contributes to low palmitic acid content phenotype Group segment), ORF, and/or cDNA.

Host cell can with comprising marking chain ORF with low palmitic acid content carrier (such as cloning vector, shuttle Carrier or expression vector) genetic engineering transformation (for example, transduction, transfection, conversion etc.).Carrier includes, such as but is not limited only to, matter Grain；Bacteriophage；Agrobacterium；Virus；Naked polynucleotides (linear or cyclic annular)；With conjugation (conjugated) polynucleotides.Perhaps Multichip carrier be directed into bacterium, especially the purpose for breeding and expanding.

Carrier can pass through any plant for being introduced into plant tissue, culture in multiple standards method known in the art It in cell and plant protoplast, including for example but is not limited only to: electroporation (From et al. (1985) Proc.Natl.Acad.Sci.USA 82:5824)；Viral vector infection, such as cauliflower mosaic virus (CaMV) is (see for example U.S. Patent number 4,407,956)；Little particle fly bomb containing nucleic acid penetrates into (ballistic penetration) (Klein et al.(1987)Nature 327:70)；Use pollen as carrier (pct international patent discloses No.WO 85/01856)；With make Agrobacterium tumefaciens or rhizobiaceae with carrying T-DNA plasmid, wherein clone has DNA fragmentation (Fraley et al. (1983)Proc.Natl.Acad.Sci.USA 80:4803).Any conjunction can be used in certain embodiments of the invention Suitable efficiently can introduce cell or the intracorporal method of plasm for nucleic acid, include but are not limited to clearly to identify herein Specific method.

Engineered host cell can be cultivated in conventional nutrient medium, or the culture by improvement Base, for example, in order to activate promoter or select transformant and in the culture medium improved.In some embodiments, host plant is thin Born of the same parents can be trained genetically modified plants.Plant regeneration is carried out from the protoplast of culture to be described in for example following document: Evans et al. (1983) " Protoplast Isolation and Culture, " is included in Handbook of Plant 124-176 pages of Co., NY, the of Cell Cultures 1, MacMillan Publishing；Davey(1983)"Recent Developments in the Culture and Regeneration of Plant Protoplasts, " it is included in 12-29 pages of Protoplasts, Birkhauser, Basel, the；Dale(1983)"Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops, " it is included in Protoplasts, ibid, the 31-41 pages；It is included in Binding (1985) " Regeneration of Plants, " 21-73 pages of Raton, FL, the of Press, Boca of Plant Protoplasts, CRC.Offer is about culture plant cell and again Other resources of raw useful details include Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems,John Wiley&Sons,Inc.,NY；Gamborg and Phillips (eds.) (1995) Plant Cell,Tissue and Organ Culture；Fundamental Methods,Springer Lab Manual, Springer-Verlag(Berlin Heidelberg NY)；With R.R.D.Croy (eds.) Plant Molecular Biology (1993)Bios Scientific Publishers,Oxford,UK(ISBN 0121983706)。

It can be cultured to regenerate using the conversion plant cell that above-mentioned any transformation technology generates and there is conversion base Because of type and therefore with the full plants of desired phenotype.These regeneration techniques commonly rely in tissue culture growth media The operation of certain plants hormone typically depends on and is introduced in intracellular kill livestock together with desired nucleotide sequence Agent and/or herbicide marker.Regeneration and cultivating process for generating full plants include the following steps: to select transformed cells And seedling；Make to convert seedling rooting；Plantlet is cultivated in the soil.

It can be used for the Plant Transformation that the nucleic acid (such as including tagger as described herein) for reducing palmitic acid content carries out Convert the species in addition to sunflower.For example, it is contemplated that contributing to or providing the low palmitic acid content phenotype in sunflower The expression product of QTL also can when being transformed into other agronomy and the important plant species of gardening and expressing wherein Enough reduce palmitic acid content.These species include dicotyledon, such as but are not limited only to, the dicotyledonous plant from following section Object: pulse family (including pea, beans (beans), lens, peanut, yam bean (yam bean), cowpea, Chenopodiaceae beans, soybean, three leaves Grass, alfalfa, lupin, vetch, lotus flower, daghestan sweet clover, Chinese wistaria and sweet pea) and composite family it is (maximum in vascular plant Section, including at least 1000 categories, including important commercial crops such as sunflower).Other include the core that can reduce palmitic acid content The plant (for example, including tagger described herein) of acid can be the plant from such as subordinate: allium, apium, Arachis, rue Tongue fur category, Capsicum, olecranon Macroptilium, Cucumis, Cucurbita, Daucus, Fagopyrum, Glycine, Helianthus, Lactuca, Lens culinaris Category, tomato genus, clover category, Pisum, Phaseolus, Solanum, Clover, Vigna and many other categories.It can be used for specific The common crop of example includes, such as but be not limited only to: soybean, sunflower, canola, pea, beans (beans) are small Hyacinth bean, peanut, yam bean, cowpea, Chenopodiaceae beans, clover, alfalfa, lupin, vetch, sweet tea clover, sweet pea, pale reddish brown pea Beans, semen viciae fabae, broccoli (broccoli), brussels sprout, wild cabbage, cauliflower (cauliflower), collard (kale), greatly Head dish (kohlrabi), celery, lettuce, carrot, onion, capsicum, potato, eggplant (eggplant) and tomato.

VIII. the system for detecting and/or being associated with low palmitic acid content label

It in some embodiments, further include containing at least one and the palmitinic acid linkage of characters low in sunflower for identifying Label plant, and/or for by the presence of specific linked marker allele system associated with low palmitic acid content, Including automated system.Example system may include the spy for detecting allele at marked locus as described herein Needle；The detector marked in detection probe；Suitable liquid handling element and temperature controller, such as mixed probe and mould Plate and/or amplification template person；And/or by label detection and expression of specific marker genes seat or allele there are associated systems Instruction.

In certain embodiments, it provides and is for identify sunflower plants of the prediction with low palmitic acid content System.This system may include, such as but be not limited only to: a group echo primer and/or probe are configured to detection and low palm At least one chain label of acid content at least one allele (such as HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and at least one chain label above-mentioned)；Detector is matched It is one or more from the output of the signal of the group echo probe or primer or its amplicon to be set to detection, identifies allele whereby Presence or absence；Refer to by the presence or absence of allele system associated with low (such as reduction) or high palmitic acid content It enables.

It executes label detection and/or associated system may include detector, be configured to detection one or more and come from The output of the signal of the group echo probe or primer or its amplicon.The concrete configuration of detector can be depended on for detecting label The type of the marker of allele.Specific example may include fluorescence detector and/or radioactive detector.For example, band The detection of the light emitting of label probe or other properties can indicate to interact (such as passing through specific hybrid) with probe The presence or absence of marker allele.Detector optionally monitors one or more signals from amplified reaction.For example, detection Device can monitor the optical signal corresponding to " real-time " amplification assay result.

Available signal detection apparatus is varied, including for example but is not limited only to, photomultiplier tube；Spectrophotometer； Ccd array；Array and Array Scanner；Scanner detector；Photoelectric tube and light emitting diode；Microscope；High-velocity scanning galvanometer (galvo-scanns)；With microfluid nucleic acid amplification detection device.In addition to the marker type for detecting marker allele Except, the concrete configuration of detector also relies partially on the instrument of user's most convenient acquisition.In some instances, it can be used Detect fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, cold light, temperature or magnetic detector.

The exact form of the instruction provided in the system according to some embodiments can be similarly varied, this is depended on The component of system.For example, instruction can be used as the system software in one or more integrated units of system exist or they It can reside in the one or more computers coupled with detector operation or in computer-readable medium.In some examples In, system command includes at least one referring to table comprising in plant or germplasm the presence or absence of specific markers allele with Association between the palmitic acid content of prediction.Instruction can also include the guidance for establishing the interface of user and system；For example, in order to Allow user to observe sample analysis result and inputs parameter into system.

In certain embodiments, system may include for for example in automation (such as full automation) system The component of the mechanized data of marker allele detected by storage or transmission description or appointment.For example, can mention For computer-readable media comprising for storing caching, main memory and the memory and/or other electronic data of computer code Storage assembly (such as hard disk drive, floppy disk drive and storage device driver).What description detected by the method for the invention The data of allele be also used as embodying (embody) computer data signal in transmission medium with electronics, light or Magnetic form is in transmission over networks, such as Intranet or internet or combinations thereof.System is acceptable or alternatively by wireless, red Outer or other available substitution transmission modes transmit data.

During operation, system generally includes sample to be analyzed, such as plant tissue or the material isolated from tissue, example Such as the genomic DNA of genomic DNA, amplification, cDNA, cDNA, RNA of amplification, amplification RNA.

In some embodiments, system can be made of isolated element, or be integrated into single unit, to facilitate inspection Marker allele is surveyed, optionally, for further executing label-phenotype association.In certain embodiments, system may be used also To include sample, such as but be not limited only to, genomic DNA from sunflower or from selected sunflower plants tissue, The genomic DNA of amplification, cDNA, cDNA, RNA of amplification, amplification RNA.

The automated system provided in some embodiments optionally includes the component for sample operation；Such as machine Device people's equipment.For example, automated system may include for solution (such as plant cell extract) to be transferred to mesh from source Ground (such as being transferred to array substrate from microwell plate) robotic liquid control bracket (armature), destination can be with number Word computer (such as in integrated computer system) operation connection.For entering data into digital computer to control The high pass quantity of fluid transfer (and arbitrarily, control is transferred on solid support by bracket) of robotic liquid control bracket Input equipment is also possible to a feature of automated system.There are many commercially available automatic machinery people liquid processing system.Example Such as, Caliper Technologies company (Hopkinton, MA) can provide a variety of automated systems, they use various Zymate^TMSystem and generally include robot and liquid treatment module.Similarly, commonRobot uses It, can also be from such as Beckman Coulter company in kinds of experiments chamber system (such as being operated for droplet price fixing) (Fullerton, CA) purchase.As a kind of substitute of Conventional robotic, Caliper Technologies and Agilent The microfluidic system for implementing liquid handling and detection that technologies (Palo Alto, CA) is produced is popularized very at present Extensively.

In certain embodiments, may include for the system of molecular marker analysis, such as, but not limited to, including high pass The digital computer of quantity of fluid control software；Number including the image analysis software for analyzing the data from label marker Word computer；Digital computer including data analysis software；For solution to be transferred to the robot liquid of destination from source Body controls bracket；For entering input into system (for example, the high pass quantity of fluid of control robotic liquid control bracket turns Move) input equipment (for example, computor-keyboard)；With for the digitized image of marker signal from labeled probe to be swept Retouch device.

By camera or other equipment (for example, photodiode and data storage device) observation and/or the optics of record Image (such as hybridization map) can be further processed in any embodiment of this paper.Such as but be not limited only to, these The processing of image, which can be, to be stored and is analyzed by image digitazation and/or on computers to image.Have perhaps commercially available More peripheral devices and the available for example various computers of software and program platform are digitized, and to digitized video or Digitized optical imagery is stored and is analyzed.

Some embodiments further include that can be used for identifying to include that at least one is chain with palmitinic acid phenotype low in sunflower The plant of label, and/or for by the presence of specific linked marker allele kit associated with low palmitic acid content. In some instances, this kit may include for detecting the suitable of at least one label chain with low palmitic acid content Primer or probe；With use these primer and probes to detect at least one label and by the palmitinic acid of marker allele and prediction The associated operation instructions of content.In some instances, kit may include for packing probe, primer and/or use The packaging material of specification；With reference substance (e.g., including the control of probe, primer or the template nucleic acid for amplification amplification is anti- Answer and molecular size marker).

In some embodiments, contain at least one mark mutually chain with palmitinic acid phenotype low in sunflower for identifying The plant of note, and/or for by the presence of specific linked marker allele kit associated with low palmitic acid content or System may include the nucleic acid for being able to detect specific SSR QTL label as described herein.For example, system or kit may include Amplimer pair can cause DNA polymerization by archaeal dna polymerase on sunflower kernel acid template to generate sunflower label Amplicon, wherein the label amplicon, which corresponds to, is selected from HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, The sunflower of HA0870, HA0907, HA0612A marks, and at least one above-mentioned mutually chain label.For example, label is special The primer pair or its equivalent that anisotropic primer pair can provide in table 6.

Embodiment

Following embodiment is provided for illustration, but is not limited only to, certain embodiments of the present invention.It should be appreciated that Embodiment described herein be only for exemplary purposes, before without departing substantially from the spirit or scope of the present invention with embodiment It puts, those skilled in the art can recognize the various reagents that can be changed, technology, system and parameter.

Embodiment 1: the natural variation of sunflower palmitic acid content

According to AOCS^TM2-66 (97) (AOCS of method Ce^TMProduct code MC-CE266) measure palmitinic acid in sunflower The natural variation of content.

5 sunflower seeds of each test sample are placed in containing 1/8 inch of (0.2825-cm) steel ball (Small Parts Inc. catalog number (Cat.No.) no.BS-0125-C) tape label 96 holes extract disk (Corning Inc. catalog number (Cat.No.) no.4411) In.200 μ L heptane are added to every hole, are then closed the lid.The sample of capping is placed in GenoGrinder^TMIn, with 1300 shake/ Minute concussion 2.0 minutes.Sample is removed, any sample not being ground is manually crushed with scoop, and regrind.

After grinding for the first time, 400 μ L heptane are added to every hole, and regrind to material with 1300 shakes/minute.Then, Sample is centrifuged 10 minutes with 3700rpm at 6 DEG C.Then, using Beckman Coulter MC robot, supernatant is shifted To with glass inserting (glass insert) (MicroLiter Analytical Supplies Inc. catalog number (Cat.No.) no.07- 8045MB-1200) and in 96 orifice plates containing 400 μ L heptane.40 μ L, 1% sodium methoxide is added to every hole.Sodium methoxide is by 30% storage Liquid is diluted with methanol (Sigma-Aldrich Fluka catalog number (Cat.No.) no.71748).Lid (mat cap) capping is padded with Teflon, and It incubates 60 minutes at room temperature before analysis.

With equipped with J&W Scientific DB-2315m x 0.25mm ID column and 0.25 μm of film thickness (Agilent Technologies, catalog no.122-2312) Agilent 6890GC-FID (Agilent Technologies) it is right Sample is analyzed, and determines that its fatty acid forms.Initial furnace temperature is 200 DEG C, and keeps the temperature during operation.Inlet set For the split ratio (split ratio) of 1:50 and 280 DEG C of temperature.Keep within initial 2 minutes the slope flow velocity of 0.8mL/ minutes helium (ramped flow rate).Then, flow velocity increased to 2.5mL/ minutes from 1.0mL/ minutes, and was kept for 1.5 minutes.Detector It is set as 300 DEG C, with constant carrier gas supplement (carrier gas make up) and 30mL/ minutes column flow rates, fuel hydrogen Flow velocity is 30mL/ minutes, and oxidant flow velocity is 400mL/ minutes.All samples are used with the volume injected of 2 μ L.

The identification of methyl hexadecanoate is by the guarantor with methyl ester reference standard (Nu-Chek-Prep, Inc., GLC#428) The time is stayed to be compared.Fig. 1 and table 1.It is that all analytes calculate individual hundred according to total mark chromatographic peak area and reference standard Divide specific area.Heptane blank is also injected, to identify any pollution on GC.

The statistics of 1. palmitic acid content of table distribution

Distribution	Quantile	% palmitinic acid
			100.0%	Maximum value	15.05
99.5		5.87
			97.5		5.23
90.0		4.63
			75.0	Quartile	4.20

50.0	Median	3.72
			25.0	Quartile	3.21
10.0		2.92
			2.5		2.66
0.5		2.41
			0.0	Minimum value	0.00

Using this method, the Sunflower Varieties developed to a part as 7 years sunflower procedure of breeding are commented The palmitic acid content of field growing sample is estimated.Fig. 2 and table 2 provide the distribution of measured palmitic acid content.Routinely to day The range of the typical observed value of palmitinic acid is the about 2.5%-6% of total fatty acids in certain herbaceous plants with big flowers germplasm, and average value is 3.75%.

The statistics of 2. palmitic acid content of table distribution

Average value	3.74945
		Standard deviation	0.70766
Standard error	0.00466
		The 95% confidence interval upper limit of average value	3.75858
95% lower limit of confidence interval of average value	3.74031

Embodiment 2: the identification of the germplasm with low palmitic acid content

It is intended at one by improveing excellent black shell, a height with the line breeding of (profile) is composed with high oleic acid In the program of linoleic acid Sunflower Lines (687R), it was found that low palmitinic acid spectrum.This is by using there is striped shell (striped- Hull), dessert (confection) parent (H280R) of high oleic acid is realized as high oleic acid donor by the method for back cross breeding 's.H280R generally has lower palmitic acid content, but observed level is usually and not less than about 2.5%.In order to Realize that target oleic levels are performed both by FAME as described in Example 1 at each during this back cross breeding program from generation to generation Analysis.In the conventional screening procedures of fatty acid levels, the segregant that palmitic acid level substantially reduces observed (segregant).In table 3, it is shown that 4 from 687R and the first backcross generations of H280R back cross breeding project individual Palmitic acid content value.

Table 3. using scheme described in embodiment 1 determine come since selecting in 687R/H280R the first back cross breeding generation The palm acid value of the sample in batch of the 8-10 grain seed of 4 floral discs (head)

Floral disc	Palmitic acid content (%)
		1	2.18
2	2.13
		3	2.06
4	2.04
		H280R	3.18

Embodiment 3: the palm fibre between low palmitinic acid parent and conventional sunflower Parents in manufactured Helianthus annuus population The variation of palmitic acid acid content

By the way that high oleic acid restorer (R system) is hybridized with the low palmitic acid origin from discovery described in embodiment 2, it was demonstrated that When excellent sunflower inbred strais hybridizes with low palmitic acid content source, palmitic acid content can morph.The low palmitic acid origin Have been inverted to the background (A system) of cytoplasmic male sterility.A system hybridizes with R system produces F₂Group, and have collected 384 Seed.Seed is cut in half, is analyzed according to half of the scheme described in embodiment 1 to seed.By the other half of seed Plantation is used for subsequent analysis.Table 4-5 provides A system/R system F₂The summary statistics of palmitic acid content in group (N=384).

F between excellent inbred strais of the table 4. with typical palmitic acid content and the strain with low palmitic acid content₂Group In palmitic acid content distribution statistics

Distribution	Quantile	% palmitinic acid
			100.0%	Maximum value	4.216
99.5		4.216
			97.5		3.612
90.0		3.342
			75.0	Quartile	3.1995
50.0	Median	2.989
			25.0	Quartile	2.5995
10.0		1.976

2.5		1.734
			0.5		1.648
0.0	Minimum value	1.648

F between excellent inbred strais of the table 5. with typical palmitic acid content and the strain with low palmitic acid content₂Group In palmitic acid content distribution statistics

Average value	2.83964
		Standard deviation	0.51878
Mean value standard error	0.02647
		The 95% confidence interval upper limit of average value	2.89169
95% lower limit of confidence interval of average value	2.78759

Embodiment 4: the proof of palmitic acid content double-peak type distribution

Fig. 3 provides the distribution of the palmitic acid content in group described in embodiment 3.Distribution is bimodal: the one of group Part concentrates on about 3.15% palmitinic acid, and low tail (lower tail) ends at about 2.5%, and high tail extends to about 4%； The second part of group concentrates on about 2.1% palmitinic acid, and low tail reaches about 1.75%, and high tail extends to about 2.5%.From The quartile that is there is provided in embodiment 3 it is observed that 25% palmitic acid content of group lower than 2.6%, group remaining Part palmitic acid content with higher.Numerical value (2.6%) the tight fits double-peak type of first quartile is distributed from lower cluster The inflection point (inflection point) of upward cluster conversion.Note that high palmitic acid content individual and low palmitic acid content individual Between have 3:1 ratio, contain it follows that there is single, main effect a genetic elements to be responsible for low palmitinic acid in the group Amount, wherein recessive allele assigns low palmitinic acid phenotype.

Embodiment 5: the QTL positioning of palmitic acid content genetic determinant

The palmitic acid content data provided using microsatellite or SSR marker and embodiment 3 and 4, by palmitic acid content Major gene resistance seat is located on sunflower linkage group 5 (LG5).

Positioning in sunflower is usually censured with linkage group.The figure of linkage group 5 is existing.See Yu et al. (2003) Crop Sci.43:367-87；See also Tang et al. (2002) Theor.Appl.Genet.105:1124-36.It should infuse Meaning, in the map developed by European Section scholar the number of sunflower linkage group with for example, above proposed in drawn bibliography Difference.Chromosome numbers corresponding with sunflower linkage group not yet define.

The primer sequence for the SSR marker for identifying palmitinic acid locus that table 6. positions on LG5 and position location

PCR program for SSR markerIn GeneAmp^TMIt is used in PCR System 9700 (Applied Biosystems) Double 384 holes module (block) carries out PCR reaction.Each PCR reaction is implemented in 8 μ L volumes, wherein containing 10ng genome DNA, the Qiagen of final concentration of 1x^TMPCR buffer (Qiagen, Valencia, California), 0.25 μM of every kind of primer (forward and reverse), 1mM MgCl₂, 0.1mM every kind of dNTP, 0.4%PVP, and 0.04 unit HotStart^TMTaq DNA polymerization Enzyme (Qiagen, Valencia, California).

PCR condition setting is as follows: 95 DEG C 12 minutes, for template DNA be denaturalized；The DNA cloning of 40 circulations (is each followed Ring: 94 DEG C are denaturalized for 5 seconds, 55 DEG C of annealing in 15 seconds and 72 DEG C of extensions in 30 seconds)；With 72 DEG C of final extensions in 30 minutes.

Fragment analysis(high pressure sterilization water will be used in the multiple final volume for being blended in 100 μ L of the PCR product of different primers pair It is supplemented to the final volume of 100 μ L).The multiple mixing PCR product of 0.5 μ L is mixed with 5 μ L sample-loading buffers.Having G5-RCT frequency Standard conditions are used on the AB3730XL DNA analysis instrument (Applied Biosystems) of spectrum matrix (spectral matrix) Run glue.Then, it imports data toIn version 4.0 (Applied Biosystems).It imports complete The dye colour in portion, and 2 highest peaks of label, minimum strength are 100 Relative fluorescence units (rfu).According to PCR fragment size Numerical value is specified to allele.Digitized allele value is imported into Excel^TM(Microsoft) in, there by them It is transformed into and is suitable for JoinMap^TM3.0 and MapQTL^TM4.0 format.

Statistical analysis

Linkage mappingUse JoinMap^TM3.0 generate A system/R system F₂The genetic linkage maps of group.JoinMap^TM3.0 wanting Ask an input file, referred to as locus gene type file.In locus gene type file, Parents allele is claimed Make " A ", donor parents allele is referred to as " B ", and the allele of heterozygote is referred to as " H ".In locus gene type file In, the data of missing are represented by dashed line.As a result it is calculated as Kosambi centimorgan.By the map generated by the analysis and public figure Spectrum (see, S.Tang, J.K.Yu, M.B.Slabaugh, D.K.Shintani, S.J.Knapp (2002) Simple sequence Repeat map of the sunflower genome.Theor.Appl.Genet.105:1124-1136) it is compared, it uses It is explained in final data.

Qtl analysisUse MapQTL^TM4.0 carry out deciding field for palmitic acid content, to position potential QTL. MapQTL^TM4.0 require 3 input files, including a locus gene type file, a map file and a quantitative data text Part.Locus gene type file contains the genotype codes of the full gene seat of segregating population as described above.Map file by JoinMap^TM3.0 generate, the figure position of the estimation containing upper 27 locus of the LG5 listed in embodiment 5.Quantitative data file Contain the palmitic acid content for using analytical chemistry methods described in embodiment 1 to determine.

Deciding field analysis assesses QTL along the probability of the deciding field between two labels.Jansen(1993) Genetics 135:205-11.Deciding field analysis is performed, and calculates the probability that QTL is located in a certain section.When When LOD score is more than scheduled P < 0.05 or the conspicuousness threshold value of P < 0.01, a QTL can be made and determine that wherein LOD score is From permutation tests (1,000iteration experiment-wise permutation test) between 1000 iteration experiments (Churchill and Doerge (1994) Genetics 138 (3): 963-71) is calculated.To have in linkage group Estimated position of the position of maximum LOD as QTL on the diagram.Since this is a F₂Group, it is therefore possible to use statistics Model carries out deciding field, to detect only QTL relevant to additive genetic variation (additive genetic variance), And using statistical models explain (therefore detect) simultaneously with additivity and dominant genetic variant (dominant genetic Variation) relevant QTL.The data of previous definition are analyzed using the two models simultaneously.

Embodiment 6: backcross progeny is selected according to palmitic acid content

Using label as described herein, to by using with allele relevant to low palmitinic acid phenotype for system The offspring for carrying out back cross breeding acquisition selects.The excellent strain that one palmitic acid content is about 3.5% has with one Low palmitinic acid phenotype is relevant etc. at locus HA0907, HA0041, HA1790, HA1665, HA0908, and HA1620 Position gene hybridizes for system.A part is selected to be returned with the excellent strain from gained offspring, to generate the first backcrossing From generation to generation.It is returned again with the excellent strain from the first backcross generations selection a part, to generate the second backcross generations.Table 7 is aobvious The genotype of the individual from the second backcross generations is shown.

7. 1, the table excellent strains with high palmitinic acid phenotype, one has equipotential base relevant to low palmitinic acid phenotype The donor of cause, and one plant of the genotype selected from using the two strains as the second backcross generations of parent and receptor

Because low palmitic acid content phenotype be it is recessive, the second backcross generations shown in table 7 individual itself will not show Show low palmitinic acid phenotype.Low palmitinic acid table can be provided under excellent background in order to verify allele relevant to low palmitinic acid Type has carried out progeny test.By the individual self-pollination in table 7, FAME points are carried out to 8 seeds for representing self-pollination progenies Analysis, to determine palmitic acid content.It is being provided in table 8 the results show that 3 in 8 offsprings have low palmitic acid content phenotype, with The desired ratio of the recessive character 1:3 controlled by term single gene seat is consistent.It is somebody's turn to do the results show that low palmitic acid content phenotype is by offspring It inherits.

The 8 of No. 19 plant self-pollination of the table 8. from the second backcross generations of ON6725R [2]/NS1982.8#1=1=3 The palmitinic acid phenotype of a single plant

Individual	Palmitic acid content (%)
		1	2.68
2	2.73
		3	1.71
4	2.23

5	1.98
		6	2.85
7	3.31
		8	1.92

Embodiment 7: selecting the cytoplasmic male sterility maintenance with high palmitic acid content is offspring

In the production of business hybrid sunflower, Cyto-plasmtc male sterile system is used to the seed of requirement. Sunflower system in embodiment 6 is a restorer, and when being used as the pollina of the female with male sterile cytoplasm, it can To restore normal fertility.It is close from the male and female for carrying the low palmitinic acid QTL allele chain with label described herein System is handed over to can produce the hybrid with low palmitic acid content phenotype, because low palmitic acid content phenotype is recessive.In cell In matter male sterility hybrid generation system, female inbred lines include two near isogenic lines: carrying and assign male sterility Cytoplasmic A system；With do not carry with normal cell matter but the B system of restoring gene.B system be it is male fertile, it can be used in It pollinates to A system, gained offspring is male sterile, because they inherit the cytoplasm from female A system.These offsprings also with A system parent is substantially the same, because A and B system is near isogene.Therefore B system, which is referred to as, keeps system.A system is to use cell from B system For matter male sterile line as donor, B system is derivative as recurrent parent.With the B system as circulation (male) parent repeatedly After backcrossing, B system genotype can be resumed, while keep the male sterile cytoplasm of donor.Resulting system is referred to as A system. The first step of the new A system-B system pair of creation one is one new B system of creation.Then, from derivative A system of B system.

In order to prove that label described herein for creating there is the cytoplasmic male sterility of low palmitic acid content phenotype to keep Be the effectiveness of purpose, by palmitic acid content be about 3.5% excellent B system in locus HA0850, HA0907 and HA0908 Locate to have with the relevant allele of low palmitinic acid phenotype for system hybridization.Remaining locus is between donor and recurrent parent It is singlet.It selects resulting offspring and the excellent system to be returned, generates the first backcross generations.Table 9 is shown from the first backcrossing The genotype of the individual of generation.

9. 1, the table excellent strain B with high palmitinic acid phenotype, one has equipotential relevant to low palmitinic acid phenotype The donor of gene, and selected from using the two strains as the first backcross generations of recurrent parent and receptor one plant Genotype

Low palm can be assigned under heterozygous state in order to verify low palmitinic acid allele entrained by the individual in table 9 Sour phenotype has carried out progeny test.By the individual self-pollination in table 9,8 seeds for representing self-pollination progenies are carried out FAME analysis, to determine palmitic acid content.It is being provided in table 10 the results show that 2 in 8 offsprings have low palmitic acid content Phenotype, it is consistent with the desired ratio of recessive character 1:3 controlled by term single gene seat.It should be the results show that low palmitic acid content table Back cross breeding gene transgression can be used into B system in type.

No. 11 plants of the table 10. from the first backcross generations of ON1919B [1] //CN1919B/NS1982.8#3=1-17=5 The individual palmitinic acid phenotype of 8 of object self-pollination

Individual	Palmitic acid content (%)
		1	2.74
2	3.16
		3	3.01
4	1.77
		5	2.97
6	3.47
		7	2.73
8	1.87

Embodiment 8: the excellent holding system of the final cytoplasmic male sterility with high palmitic acid content and restorer are developed

After backcrossing two instead of, by selected 3 generation of individual self-pollination, choosing, there is desired Agronomic character person to carry out FAME Analysis.The results show that can be developed using back cross breeding method with the final excellent of low palmitic acid content phenotype shown in table 11 Good B system.

The palmitic acid content for the excellent B strain that table 11. is developed using back cross breeding method

Title	Palmitic acid content (%)
		H251B [3]/NS1982.12-20=1=4-1-20-07	1.93
ON7479B [2]/NS1982-8#2=1=5-12-10-01	1.96

After backcrossing two instead of, by selected individual 3 generation of self-pollination, there is desired Agronomic character person to carry out FAME points for selection Analysis.The results show that can be developed using back cross breeding method with the final excellent of low palmitic acid content phenotype shown in table 12 Restorer.

The palmitic acid content for the Elite restorer line that table 12. is developed using back cross breeding method

Title	Palmitic acid content (%)
		OND163R [4]/NS1982-16=20=2=3=13-7-2-2	1.79
ON7385R [3]/NS1982.8=7=2=5-4-2-01	1.79

Although foregoing embodiments have been described in detail for the purpose for illustrating and understanding, this field Technical staff by read the disclosure can it is obvious under the premise of without departing substantially from true scope of the present invention in form and details Various changes.For example, above-mentioned all technology and equipments can according to it is various combination be subject to using.

Claims (19)

1. a kind of method for identifying the first sunflower plants or germplasm comprising low palmitic acid content, this method comprises:

Detect at least one of the first sunflower plants or germplasm label chain with low palmitic acid content, wherein this at least one Kind label is selected from HA0612A,

When detecting at least one label amplicon, identifies first sunflower plants or germplasm includes low palmitic acid content； With

The first sunflower plants for identifying low palmitic acid content or germplasm are hybridized with the second sunflower plants or germplasm, are generated Sunflower plants comprising low palmitic acid content or germplasm offspring.

2. the method according to claim 1, wherein the detection includes that detection polymorphism simple sequence repeats mark (SSR) extremely A few allelic form.

3. the method according to claim 1, wherein the detection includes expanding a part of the label or the label and producing The raw label amplicon through expanding, and the resulting label amplicon through expanding of detection.

4. according to the method in claim 3, wherein the amplification includes:

The nucleic acid that amplimer or amplimer pair are separated with from first sunflower plants or germplasm mixes, and wherein this draws Object or primer pair is complementary at least part of the label or partial complementarity, and the sunflower nucleic acid is able to use as template Cause DNA caused by archaeal dna polymerase to polymerize；With

Extend the primer or primer pair in the DNA polymerization reaction comprising archaeal dna polymerase and template nucleic acid, to generate at least One amplicon.

5. method according to claim 4, wherein the nucleic acid is DNA molecular or RNA molecule.

6. according to the method in claim 3, wherein the amplification is including the use of polymerase chain reaction (PCR) or ligase chain type It reacts (LCR), uses the nucleic acid separated from first sunflower plants or germplasm as template in PCR or LCR.

7. the method according to claim 1, wherein the detected label is determined with software selected from the group below: TASSEL^TM,GeneFlow^TM, and MapManager-QTX^TM。

8. the method according to claim 1, wherein this method includes that will represent the data electron-transport or electricity of detected label Son is stored in computer-readable medium.

9. the method according to claim 1, wherein this method includes the offspring of selection first sunflower plants or germplasm.

10. a kind of method for generating the genetically modified plants with low palmitic acid content phenotype, this method comprises:

One or more exogenous nucleic acids are imported to generate genetically modified plants in target plant, wherein the one or more external source At least one of nucleic acid includes the endogenous nucleotide sequences from sunflower plants, at least one in the sunflower plants The label of a selection the following group is chain: HA0612A,

Wherein, resulting genetically modified plants show reduced palmitic acid content.

11. method according to claim 10, wherein the target plant is sunflower plants.

12. method according to claim 10, wherein the endogenous nucleotide sequences from sunflower plants are in the sunflower It is chain at least one described label in plant so that the endogenous nucleotide sequences show with it is described at least one label do not surpass Cross 10% genetic recombination frequency.

13. method according to claim 10, wherein the nucleotide sequence is separated by positional cloning, and by with institute State the chain and identified of at least one label.

14. method according to claim 10, wherein the exogenous nucleic acid sequences correspond to the open reading frame of coding polypeptide (ORF), the polypeptide causes the sunflower plants to have reduced palmitic acid content when expressing in sunflower plants.

15. method according to claim 10, wherein the exogenous nucleic acid includes expression vector.

16. a kind of for identifying that the system for being predicted to have the sunflower plants of low palmitic acid content phenotype, the system include:

One group echo probe or primer are configured to detect at least one label chain with low palmitic acid content, wherein the mark Note is selected from HA0612A；

Detector, is configured to detect and exports from one or more signals of the label probe or primer sets or its amplicon, by This identifies the existence or non-existence of the label；With

By the existence or non-existence and the low associated system command of palmitic acid content phenotype of the label.

17. the system of claim 16, wherein the detector detects one or more light emittings, the wherein light emitting indicateing arm Remember the existence or non-existence of allele.

18. the system of claim 16, wherein described instruction includes at least one referring to table, it is described referring to table include it is described at least The marker allele of one detected mark existence or non-existence with being associated between low palmitic acid content phenotype.

19. the system of claim 16, wherein the system includes the nucleic acid samples from plant or germplasm.